Analytical Chemistry

BRYAN M. HAM, Ph.D., has worked in analytical chemistry laboratories for over 25 years including petroleum, chemical, environmental, foodstuff, and life science research, and has a doctorate in analytical chemistry. He has published 15 research papers in peer reviewed journals and two books: Even Electron Mass Spectrometry with Biomolecule Applications (Wiley, 2008), and Proteomics of Biological Systems: Protein Phosphorylation Using Mass Spectrometry Techniques (Wiley, 2012). He is currently working for the Department of Homeland Security at the U.S. Customs and Border Protection New York Laboratory. He is a member of the American Society of Mass Spectrometry (ASMS) and the American Chemical Society (ACS). AIHUI MAHAM, Ph.D., is an expert in nano-materials including the synthesis and characterization of chemical and biological nano-sensors. She is also an expert in the field of inorganic materials chemistry, and their characterization utilizing methodologies such as SEM, XRD, XRF and OES. She has published numerous research papers including a recent review entitled Protein-Based Nanomedicine Platforms for Drug Delivery (Small, 2009), which has been cited over 170 times by other researchers. She is currently working for the Department of Homeland Security at the U.S. Customs and Border Protection New York Laboratory.

Preface xxiii

Author Biographies xxv

Acknowledgments xxvii

1 Chemist and Technician in the Analytical Laboratory 1

1.1 Introduction—The Analytical Chemist and Technician 1

1.2 Today’s Laboratory Chemist and Technician 1

1.3 ChemTech—The Chemist and Technician Toolkit Companion 1

1.4 Chapter Layout 2

1.5 Users of ChemTech 6

2 Introduction to the Analytical Laboratory 7

2.1 Introduction to the Laboratory 7

2.2 Laboratory Glassware 7

2.3 Conclusion 18

3 Laboratory Safety 19

3.1 Introduction 19

3.2 Proper Personal Protection and Appropriate Attire 19

3.3 Proper Shoes and Pants 20

3.4 Laboratory Gloves 20

3.5 General Rules to Use Gloves 22

3.6 Material Safety Data Sheet (MSDS) 22

3.7 Emergency Eye Wash and Face Wash Stations 23

3.8 Emergency Safety Showers 24

3.9 Fire Extinguishers 24

3.10 Clothing Fire in the Laboratory 25

3.11 Spill Cleanup Kits 25

3.12 Chemicals and Solvents 27

3.13 First Aid Kits 27

3.14 Gasses and Cylinders 29

3.15 Sharps Containers and Broken Glass Boxes 29

3.16 Occupational Safety and Health Administration (OSHA) 29

4 Basic Mathematics in the Laboratory 83

4.1 Introduction to Basic Math 83

4.2 Units and Metric System 83

4.3 Significant Figures 84

4.4 Scientific Calculators 86

4.5 ChemTech Conversion Tool 89

4.6 Chapter Key Concepts 89

4.7 Chapter Problems 92

5 Analytical Data Treatment (Statistics) 93

5.1 Errors in the Laboratory 93

5.2 Expressing Absolute and Relative Errors 94

5.3 Precision 94

5.4 The Normal Distribution Curve 94

5.5 Precision of Experimental Data 96

5.6 Normal Distribution Curve of a Sample 97

5.7 ChemTech Statistical Calculations 98

5.8 Student’s Distribution t Test for Confidence Limits 101

5.9 Tests of Significance 104

5.10 Treatment of Data Outliers 105

5.11 Chapter Key Concepts 106

5.12 Chapter Problems 107

6 Plotting and Graphing 109

6.1 Introduction to Graphing 109

6.2 Graph Construction 109

6.3 Rectangular Cartesian Coordinate System 110

6.4 Curve Fitting 110

6.5 Redrawn Graph Example 110

6.6 Graphs of Equations 111

6.7 Least-Squares Method 114

6.8 Computer-Generated Curves 115

6.9 Calculating Concentrations 119

6.10 Nonlinear Curve Fitting 119

6.11 Chapter Key Concepts 123

6.12 Chapter Problems 124

7 Using Microsoft Excel® in the Laboratory 125

7.1 Introduction to Excel® 125

7.2 Opening Excel® in ChemTech 125

7.3 The Excel® Spreadsheet 125

7.4 Graphing in Excel® 127

7.5 Charts in Excel® 2010 132

7.6 Complex Charting in Excel® 97-2003 132

7.7 Complex Charting in Excel® 2010 139

7.8 Statistical Analysis Using Excel® 141

8 Making Laboratory Solutions 147

8.1 Introduction 147

8.2 Laboratory Reagent Fundamentals 147

8.3 The Periodic Table 147

8.4 Calculating Formula Weights 148

8.5 Calculating the Mole 148

8.6 Molecular Weight Calculator 148

8.7 Expressing Concentration 148

8.8 The Parts per (PP) Notation 153

8.9 Computer-Based Solution Calculations 153

8.10 Reactions in Solution 157

8.11 Chapter Key Concepts 157

8.12 Chapter Problems 158

9 Acid–Base Theory and Buffer Solutions 159

9.1 Introduction 159

9.2 Acids and Bases in Everyday Life 159

9.3 The Litmus Test 159

9.4 Early Acid–Base Descriptions 160

9.5 Brǿnsted–Lowry Definition 160

9.6 The Equilibrium Constant 161

9.7 The Acid Ionization Constant 161

9.8 Calculating the Hydrogen Ion Concentration 162

9.9 The Base Ionization Constant 163

9.10 Ion Product for Water 164

9.11 The Solubility Product Constant (Ksp) 164

9.12 The pH of a Solution 166

9.13 Measuring the pH 167

9.14 Buffered Solutions—Description and Preparing 168

9.15 ChemTech Buffer Solution Calculator 170

9.16 Chapter Key Concepts 171

9.17 Chapter Problems 172

10 Titration—A Volumetric Method of Analysis 175

10.1 Introduction 175

10.2 Reacting Ratios 175

10.3 The Equivalence Point 176

10.4 Useful Relationships for Calculations 176

10.5 Deriving the Titration Equation 176

10.6 Titrations in ChemTech 177

10.7 Acid/Base Titration Endpoint (Equivalence Point) 178

10.8 Acid/Base Titration Midpoint 179

10.9 Acid/Base Titration Indicators 180

10.10 Titrations Using Normal Solutions 181

10.11 Polyprotic Acid Titration 181

10.12 ChemTech Calculation of Normal Titrations 182

10.13 Performing a Titration 183

10.14 Primary Standards 184

10.15 Standardization of Sodium Hydroxide 185

10.16 Conductometric Titrations (Nonaqueous Solutions) 186

10.17 Precipitation Titration (Mohr Method for Halides) 188

10.18 Complex Formation with Back Titration (Volhard Method for Anions) 189

10.19 Complex Formation Titration with EDTA for Cations 190

10.20 Chapter Key Concepts 194

10.21 Chapter Problems 195

11 Oxidation–Reduction (Redox) Reactions 197

11.1 Introduction 197

11.2 Oxidation and Reduction 197

11.3 The Volt 198

11.4 The Electrochemical Cell 198

11.5 Redox Reaction Conventions 198

11.6 The Nernst Equation 200

11.7 Determining Redox Titration Endpoints 202

11.8 Potentiometric Titrations 202

11.9 Visual Indicators Used in Redox Titrations 204

11.10 Pretitration Oxidation–Reduction 205

11.11 Ion-Selective Electrodes 206

11.12 Chapter Key Concepts 206

11.13 Chapter Problems 207

12 Laboratory Information Management System (LIMS) 209

12.1 Introduction 209

12.2 LIMS Main Menu 209

12.3 Logging in Samples 209

12.4 Entering Test Results 209

12.5 Add or Delete Tests 211

12.6 Calculations and Curves 212

12.7 Search Wizards 214

12.8 Approving Samples 218

12.9 Printing Sample Reports 220

13 Ultraviolet and Visible (UV/Vis) Spectroscopy 221

13.1 Introduction to Spectroscopy in the Analytical Laboratory 221

13.2 The Electromagnetic Spectrum 221

13.3 Ultraviolet/Visible (UV/Vis) Spectroscopy 221

13.4 UV/Visible Spectrophotometers 230

13.5 Special Topic (Example)—Spectrophotometric Study of Dye Compounds 234

13.6 Chapter Key Concepts 236

13.7 Chapter Problems 237

14 Fluorescence Optical Emission Spectroscopy 239

14.1 Introduction to Fluorescence 239

14.2 Fluorescence and Phosphorescence Theory 240

14.3 Phosphorescence 241

14.4 Excitation and Emission Spectra 242

14.5 Rate Constants 243

14.6 Quantum Yield Rate Constants 243

14.7 Decay Lifetimes 244

14.8 Factors Affecting Fluorescence 244

14.9 Quantitative Analysis and Beer–Lambert Law 248

14.10 Quenching of Fluorescence 249

14.11 Fluorometric Instrumentation 249

14.12 Special Topic—Flourescence Study of Dye-A007 Complexes 255

14.13 Chapter Key Concepts 257

14.14 Chapter Problems 258

15 Fourier Transform Infrared (FTIR) Spectroscopy 261

15.1 Introduction 261

15.2 Basic IR Instrument Design 261

15.3 The Infrared Spectrum and Molecular Assignment 263

15.4 FTIR Table Band Assignments 264

15.5 FTIR Spectrum Example I 270

15.6 FTIR Spectrum Example II 270

15.7 FTIR Inorganic Compound Analysis 271

15.8 Chapter Key Concepts 271

15.9 Chapter Problems 273

16 Nuclear Magnetic Resonance (NMR) Spectroscopy 277

16.1 Introduction 277

16.2 Frequency and Magnetic Field Strength 277

16.3 Continuous-Wave NMR 278

16.4 The NMR Sample Probe 280

16.5 Pulsed Field Fourier Transform NMR 280

16.6 Proton NMR Spectra Environmental Effects 280

16.7 Carbon-13 NMR 283

16.8 Special Topic—NMR Characterization of Cholesteryl Phosphate 287

16.9 Chapter Key Concepts 292

16.10 Chapter Problems 293

References 294

17 Atomic Absorption Spectroscopy (AAS) 295

17.1 Introduction 295

17.2 Atomic Absorption and Emission Process 295

17.3 Atomic Absorption and Emission Source 296

17.4 Source Gases and Flames 296

17.5 Block Diagram of AAS Instrumentation 296

17.6 The Light Source 297

17.7 Interferences in AAS 299

17.8 Electrothermal Atomization—Graphite Furnace 299

17.9 Instrumentation 300

17.10 Flame Atomic Absorption Analytical Methods 301

18 Atomic Emission Spectroscopy 303

18.1 Introduction 303

18.2 Elements in Periodic Table 303

18.3 The Plasma Torch 303

18.4 Sample Types 304

18.5 Sample Introduction 304

18.6 ICP-OES Instrumentation 305

18.7 ICP-OES Environmental Application Example 310

19 Atomic Mass Spectrometry 325

19.1 Introduction 325

19.2 Low-Resolution ICP-MS 325

19.3 High-Resolution ICP-MS 328

20 X-ray Fluorescence (XRF) and X-ray Diffraction (XRD) 333

20.1 X-Ray Fluorescence Introduction 333

20.2 X-Ray Fluorescence Theory 333

20.3 Energy-Dispersive X-Ray Fluorescence (EDXRF) 334

20.4 Wavelength Dispersive X-Ray Fluorescence (WDXRF) 337

20.5 Applications of XRF 341

20.6 X-ray Diffraction (XRD) 342

21 Chromatography—Introduction and Theory 351

21.1 Preface 351

21.2 Introduction to Chromatography 351

21.3 Theory of Chromatography 351

21.4 The Theoretical Plate Number N 355

21.5 Resolution RS 356

21.6 Rate Theory versus Plate Theory 357

21.7 Retention Factor k 361

References 362

22 High Performance Liquid Chromatography (HPLC) 363

22.1 HPLC Background 363

22.2 Design and Components of HPLC 363

23 Solid-Phase Extraction 381

23.1 Introduction 381

23.2 Disposable SPE Columns 381

23.3 SPE Vacuum Manifold 381

23.4 SPE Procedural Bulletin 381

24 Plane Chromatography: Paper and Thin-Layer Chromatography 395

24.1 Plane Chromatography 395

24.2 Thin-Layer Chromatography 395

24.3 Retardation Factor (RF) in TLC 398

24.4 Plate Heights (H) and Counts (N) in TLC 398

24.5 Retention Factor in TLC 399

25 Gas-Liquid Chromatography 401

25.1 Introduction 401

25.2 Theory and Principle of GC 401

25.3 Mobile-Phase Carrier Gasses in GC 403

25.4 Columns and Stationary Phases 404

25.5 Gas Chromatograph Injection Port 406

25.6 The GC Oven 415

25.7 GC Programming and Control 417

25.8 GC Detectors 418

26 Gas Chromatography–Mass Spectrometry (GC–MS) 421

26.1 Introduction 421

26.2 Electron Ionization (EI) 421

26.3 Electron Ionization (EI)/OE Processes 422

26.4 Oleamide Fragmentation Pathways: OE M+• by Gas Chromatography/Electron Ionization Mass Spectrometry 425

26.5 Oleamide Fragmentation Pathways: EE [M+H]+ by ESI/Ion Trap Mass Spectrometry 426

26.6 Quantitative Analysis by GC/EI–MS 429

26.7 Chapter Problems 431

References 433

27 Special Topics: Strong Cation Exchange Chromatography and Capillary Electrophoresis 435

27.1 Introduction 435

27.2 Strong Ion Exchange HPLC 435

27.3 CZE 435

27.4 Binding Constants by Cation Exchange and CZE 436

27.5 Comparison of Methods 446

27.6 Conclusions 448

References 448

28 Mass Spectrometry 449

28.1 Definition and Description of Mass Spectrometry 449

28.2 Basic Design of Mass Analyzer Instrumentation 449

28.3 Mass Spectrometry of Protein Metabolite and Lipid Biomolecules 451

28.4 Fundamental Studies of Biological Compound Interactions 455

28.5 Mass-to-Charge (m/z) Ratio: How the Mass Spectrometer Separates Ions 457

28.6 Exact Mass versus Nominal Mass 458

28.7 Mass Accuracy and Resolution 460

28.8 High-Resolution Mass Measurements 461

28.9 Rings Plus Double Bonds (r + db) 463

28.10 The Nitrogen Rule in Mass Spectrometry 464

28.11 Chapter Problems 465

References 465

29 Ionization in Mass Spectrometry 467

29.1 Ionization Techniques and Sources 467

29.2 Chemical Ionization (CI) 467

29.3 Atmospheric Pressure Chemical Ionization (APCI) 471

29.4 Electrospray Ionization (ESI) 472

29.5 Nanoelectrospray Ionization (Nano-ESI) 474

29.6 Atmospheric Pressure Photo Ionization (APPI) 477

29.7 Matrix Assisted Laser Desorption Ionization (MALDI) 483

29.8 FAB 485

29.9 Chapter Problems 489

References 489

30 Mass Analyzers in Mass Spectrometry 491

30.1 Mass Analyzers 491

30.2 Magnetic and Electric Sector Mass Analyzer 491

30.3 Time-of-Flight Mass Analyzer (TOF/MS) 496

30.4 Time-of-Flight/Time-of-Flight Mass Analyzer (TOF–TOF/MS) 497

30.5 Quadrupole Mass Filter 500

30.6 Triple Quadrupole Mass Analyzer (QQQ/MS) 502

30.7 Three-Dimensional Quadrupole Ion Trap Mass Analyzer (QIT/MS) 503

30.8 Linear Quadrupole Ion Trap Mass Analyzer (LTQ/MS) 506

30.9 Quadrupole Time-of-Flight Mass Analyzer (Q-TOF/MS) 507

30.10 Fourier Transform Ion Cyclotron Resonance Mass Analyzer (FTICR/MS) 508

30.11 Linear Quadrupole Ion Trap Fourier Transform Mass Analyzer (LTQ–FT/MS) 517

30.12 Linear Quadrupole Ion Trap Orbitrap Mass Analyzer (LTQ–Orbitrap/MS) 518

30.13 Chapter Problems 527

References 527

31 Biomolecule Spectral Interpretation: Small Molecules 529

31.1 Introduction 529

31.2 Ionization Efficiency of Lipids 529

31.3 Fatty Acids 530

31.4 Wax Esters 537

31.5 Sterols 542

31.6 Acylglycerols 548

31.7 ESI-Mass Spectrometry of Phosphorylated Lipids 551

31.8 Chapter Problems 556

References 557

32 Macromolecule Analysis 559

32.1 Introduction 559

32.2 Carbohydrates 559

32.3 Nucleic Acids 565

32.4 Chapter Problems 576

References 577

33 Biomolecule Spectral Interpretation: Proteins 579

33.1 Introduction to Proteomics 579

33.2 Protein Structure and Chemistry 579

33.3 Bottom-up Proteomics: Mass Spectrometry of Peptides 580

33.4 Top-Down Proteomics: Mass Spectrometry of Intact Proteins 590

33.5 PTM of Proteins 594

33.6 Systems Biology and Bioinformatics 614

33.7 Chapter Problems 618

References 619

Appendix I: Chapter Problem Answers 621

Appendix II: Atomic Weights and Isotopic Compositions 627

Appendix III: Fundamental Physical Constants 631

Appendix IV: Redox Half Reactions 633

Appendix V: Periodic Table of Elements 637

Appendix VI: Installing and Running Programs 639

Index 641