Bioanalytical Chemistry

Bioanalytical ChemistrySecond Edition

Susan R. MikkelsenEduardo Cortón

Copyright 2016 by John Wiley & Sons, Inc. All rights reserved.

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Library of Congress Cataloging-in-Publication Data:

Names: Mikkelsen, Susan R., 1960- | Cortón, Eduardo, 1962­Title: Bioanalytical chemistry / Susan R. Mikkelsen, Eduardo Cortón.Description: Second edition. | Hoboken, New Jersey : John Wiley & Sons, Inc.,

[2016] | Includes index.Identifiers: LCCN 2015041460 (print) | LCCN 2015043820 (ebook) | ISBN 9781118302545 (cloth) |

ISBN 9781119057703 (pdf) | ISBN 9781119057635 (epub)Subjects: LCSH: Analytical biochemistry. | Chemistry, Analytic.Classification: LCC QP519.7 .M54 2016 (print) | LCC QP519.7 (ebook) | DDC

612/.01585–dc23LC record available at http://lccn.loc.gov/2015041460

Printed in the United States of America.

10 9 8 7 6 5 4 3 2 1

Contents

Preface to Second Edition xix

Preface to First Edition xxi

Acknowledgments xxiii

1. Quantitative Instrumental Measurements 1

1.1. Introduction 11.2. Optical Measurements 2

1.2.1. UV-Visible Absorbance 31.2.2. Turbidimetry (Light-Scattering) 51.2.3. Fluorescence 51.2.4. Chemiluminescence and Bioluminescence 7

1.3. Electrochemical Measurements 8

1.3.1. Potentiometry 101.3.2. Amperometry 101.3.3. Impedimetry 11

1.4. Radiochemical Measurements 12

1.4.1. Scintillation Counting 121.4.2. Geiger Counting 12

1.5. Surface Plasmon Resonance 131.6. Calorimetry 14

1.6.1. Differential Scanning Calorimetry (DSC) 151.6.2. Isothermal Titration Calorimetry (ITC) 16

1.7. Automation: Microplates, Multiwell Liquid Dispensers and MicroplateReaders 16

1.8. Calibration of Instrumental Measurements 18

1.8.1. External Standards 181.8.2. Internal Standards 191.8.3. Standard Additions 20

v

vi Contents

1.9. Quantitative and Semi-Quantitative Measurements 21

1.9.1. Exact Concentration 211.9.2. Positive or Negative Result 21

Suggested Reading 22Problems 22

2. Spectroscopic Methods for the Quantitation of Classes of Biomolecules 23

2.1. Introduction 232.2. Total Protein 24

2.2.1. Lowry Method 242.2.2. Smith (BCA) Method 242.2.3. Bradford Method 262.2.4. Ninhydrin-Based Assay 272.2.5. Other Protein Quantitation Methods 28

2.3. Total DNA 31

2.3.1. Diaminobenzoic Acid (DABA) Method 322.3.2. Diphenylamine (DPA) Method 322.3.3. Other Fluorimetric Methods 33

2.4. Total RNA 342.5. Total Carbohydrate 35

2.5.1. Ferricyanide Method 352.5.2. Phenol-Sulfuric Acid Method 362.5.3. 2-Aminothiophenol Method 362.5.4. Purpald Assay for Bacterial Polysaccharides 37

2.6. Free Fatty Acids 37

References 38Problems 39

3. Enzymes

3.1. Introduction 413.2. Enzyme Nomenclature 423.3. Enzyme Commission Numbers 433.4. Enzymes in Bioanalytical Chemistry 453.5. Enzyme Kinetics 46

3.5.1. Simple One-Substrate Enzyme Kinetics 483.5.2. Experimental Determination of Michaelis-Menten

Parameters 50

3.5.2.1. Eadie-Hofstee Method 503.5.2.2. Hanes Method 50

41

Contents vii

3.5.2.3. Lineweaver-Burk Method 513.5.2.4. Cornish-Bowden-Eisenthal Method 52

3.5.3. Comparison of Methods for the Determination of KM Values 523.5.4. One-Substrate, Two-Product Enzyme Kinetics 543.5.5. Two-Substrate Enzyme Kinetics 543.5.6. Examples of Enzyme-Catalyzed Reactions and their

Treatment 563.5.7. Curve Fitting for Enzyme Kinetic Data 57

3.6. Enzyme Activators 583.7. Enzyme Inhibitors 59

3.7.1. Competitive Inhibition 603.7.2. Noncompetitive Inhibition 603.7.3. Uncompetitive Inhibition 62

3.8. Enzyme Units and Concentrations 62

Suggested Reading 64References 64Problems 64

4. Quantitation of Enzymes and Their Substrates 67

4.1. Introduction 674.2. Substrate Depletion or Product Accumulation 684.3. Direct and Coupled Measurements 694.4. Classification of Methods 714.5. Instrumental Methods 73

4.5.1. Optical Detection 73

4.5.1.1. Absorbance 734.5.1.2. Fluorescence 754.5.1.3. Luminescence 774.5.1.4. Nephelometry 79

4.5.2. Electrochemical Detection 79

4.5.2.1. Amperometry 794.5.2.2. Potentiometry 804.5.2.3. Conductimetry 80

4.5.3. Other Detection Methods 81

4.5.3.1. Radiochemical 814.5.3.2. Manometry 814.5.3.3. Calorimetry 82

4.6. High-Throughput Assays for Enzymes and Inhibitors 82

viii Contents

4.7. Assays for Enzymatic Reporter Gene Products 844.8. Practical Considerations for Enzymatic Assays 85

Suggested Reading 86References 86Problems 87

5. Immobilized Enzymes 90

5.1. Introduction 905.2. Immobilization Methods 90

5.2.1. Nonpolymerizing Covalent Immobilization 91

5.2.1.1. Controlled-Pore Glass 925.2.1.2. Polysaccharides 935.2.1.3. Polyacrylamide 955.2.1.4. Acidic Supports 955.2.1.5. Anhydride Groups 965.2.1.6. Thiol Groups 97

5.2.2. Crosslinking with Bifunctional Reagents 975.2.3. Adsorption 985.2.4. Entrapment 995.2.5. Microencapsulation 100

5.3. Properties of Immobilized Enzymes 1015.4. Immobilized Enzyme Reactors 1075.5. Theoretical Treatment of Packed-Bed Enzyme Reactors 109

Suggested Reading 113References 113Problems 114

6. Antibodies 117

6.1. Introduction 1176.2. Structural and Functional Properties of Antibodies 1186.3. Polyclonal and Monoclonal Antibodies 1216.4. Antibody-Antigen Interactions 1226.5. Analytical Applications of Secondary Antibody-Antigen

Interactions 124

6.5.1. Agglutination Reactions 1246.5.2. Precipitation Reactions 126

Suggested Reading 129References 129Problems 129

Contents ix

7. Quantitative Immunoassays with Labels 131

7.1. Introduction 1317.2. Labeling Reactions 1327.3. Heterogeneous Immunoassays 134

7.3.1. Labeled-Antibody Methods 1367.3.2. Labeled-Ligand Assays 1367.3.3. Radioisotopes 1397.3.4. Fluorophores 139

7.3.4.1. Indirect Fluorescence 1407.3.4.2. Competitive Fluorescence 1407.3.4.3. Sandwich Fluorescence 1407.3.4.4. Fluorescence Excitation Transfer 1407.3.4.5. Time-Resolved Fluorescence 141

7.3.5. Quantum Dots 1427.3.6. Chemiluminescent Labels 1437.3.7. Enzyme Labels 1457.3.8. Lateral Flow Immunoassay 148

7.4. Homogeneous Immunoassays 149

7.4.1. Fluorescent Labels 149

7.4.1.1. Enhancement Fluorescence 1497.4.1.2. Direct Quenching Fluorescence 1507.4.1.3. Indirect Quenching Fluorescence 1507.4.1.4. Fluorescence Polarization Immunoassay 1517.4.1.5. Fluorescence Excitation Transfer 151

7.4.2. Enzyme Labels 152

7.4.2.1. Enzyme-Multiplied Immunoassay Technique 1527.4.2.2. Substrate-Labelled Fluorescein Immunoassay 1537.4.2.3. Apoenzyme Reactivation Immunoassay 1537.4.2.4. Cloned Enzyme Donor Immunoassay 1547.4.2.5. Enzyme Inhibitory Homogeneous Immunoassay 154

7.5. Evaluation of New Immunoassay Methods 155

Suggested Reading 160References 160Problems 161

8. Biosensors 166

8.1. Introduction 1668.2. Biosensor Diversity and Classification 1698.3. Recognition Agents 171

x Contents

8.3.1. Natural Recognition Agents 1718.3.2. Artificial Recognition Agents 172

8.4. Response of Enzyme-Based Biosensors 1758.5. Examples of Biosensor Configurations 178

8.5.1. Ferrocene-Mediated Amperometric Glucose Sensor 1788.5.2. Potentiometric Biosensor for Phenyl Acetate 1808.5.3. Evanescent-Wave Fluorescence Biosensor for

Bungarotoxin 1818.5.4. Optical Biosensor for Glucose Based on Fluorescence Resonance

Energy Transfer 1838.5.5. Piezoelectric Sensor for Nucleic Acid Detection 1848.5.6. Enzyme Thermistors 1868.5.7. Fluorescence Sensor for Nitroaromatic Explosives Based on a

Molecularly Imprinted Polymer 1878.5.8. Immunosensor Microwell Arrays from Gold Compact

Disks 1888.5.9. Nanoparticle-Enhanced Detection of Thrombin by SPR 1908.5.10. Environmental BOD and Toxicity Biosensors Based on Viable

Cells 1928.5.11. Detection of Viruses using a Surface Acoustic Wave (SAW)

Biosensor 1938.5.12. MEMS Microcantilever Biosensor for Virus Detection 1968.5.13. DNA Microarrays 198

8.6. Evaluation of Biosensor Perfomance 2018.7. In Vivo Applications of Biosensors 202

8.7.1. Biocompatible Materials 2038.7.2. Physiological Environment of the Human Body 2038.7.3. The Artificial Pancreas 2058.7.4. An Enzymatic Fuel Cell as a Component of an Implanted

Biosensing System 2058.7.5. Other Examples of Implantable Biosensors 206

Suggested Reading 207References 207Problems 209

9. Directed Evolution for the Design of Macromolecular Reagents

9.1. Introduction 2109.2. Rational Design and Directed Evolution 2119.3. Generation of Genetic Diversity 214

9.3.1. Polymerase Chain Reaction and Error-Prone PCR 2159.3.2. DNA Shuffling 217

210

Contents xi

9.4. Linking Genotype and Phenotype 217

9.4.1. Cell Expression and Cell Surface Display (In vivo) 2189.4.2. Phage Display (In vivo) 2189.4.3. Ribosome Display (In vitro) 2199.4.4. mRNA-Peptide Fusion (In vitro) 2209.4.5. Microcompartmentalization (In vitro) 220

9.5. Identification and Selection of Successful Variants 221

9.5.1. Identification of Successful Variants Based on BindingProperties 222

9.5.2. Identification of Successful Variants Based on CatalyticActivity 222

9.6. Examples of Directed Evolution Experiments 224

9.6.1. Directed Evolution of Galactose Oxidase 2249.6.2. α-Hemolysin Evolution 225

Suggested Reading 226References 226Problems 227

10. Image-Based Bioanalysis

10.1. Introduction 22910.2. Magnification and Resolution 23010.3. Optical Microscopy 231

10.3.1. The Compound Light Microscope 23110.3.2. The Confocal Microscope 23110.3.3. Sample Preparation 23210.3.4. General and Selective Stains 23310.3.5. Fluorescence In situ Hybridization 23410.3.6. Green Fluorescent Protein and its Analogues 234

10.4. Electron Microscopy 234

10.4.1. Principles and Instrumentation 23410.4.2. Sample Preparation 23510.4.3. Transmission Electron Microscopy (TEM) 23610.4.4. Scanning Electron Microscopy (SEM) 236

10.5. Scanning Tunneling Microscopy 237

10.5.1. Principles and Instrumentation 23710.5.2. Biological Applications 237

10.6. Atomic Force Microscopy (AFM) 237

10.6.1. Cantilevers and Operational Modes 23710.6.2. Samples and Substrates 239

229

xii Contents

10.6.3. Biological Applications 23910.6.4. Four-Dimensional (4D) Scanning 240

10.7. Scanning Electrochemical Microscopy (SECM) 240

10.7.1. Principles and Instrumentation 24010.7.2. Samples and Substrates 24110.7.3. Biological Applications 241

Suggested Reading 242References 242Problems 243

11.Principles of Electrophoresis

11.1. Introduction 24411.2. Electrophoretic Support Media 248

11.2.1. Paper 24811.2.2. Starch Gels 24911.2.3. Polyacrylamide Gels 25011.2.4. Agarose Gels 25411.2.5. Polyacrylamide-Agarose Gels 254

11.3. Effect of Experimental Conditions Onelectrophoretic Separations 25411.4. Electric Field Strength Gradients 25511.5. Pulsed Field Gel Electrophoresis (PFGE) 25611.6. Detection of Proteins and Nucleic Acids After Electrophoretic

Separation 258

11.6.1. Stains and Dyes 25811.6.2. Detection of Enzymes by Substrate Staining 26011.6.3. The Southern Blot 26011.6.4. The Northern Blot 26211.6.5. The Western Blot 26211.6.6. Detection of DNA Fragments on Membranes with DNA

Probes 263

Suggested Reading 265References 266Problems 266

12.Applications of Zone Electrophoresis 268

12.1. Introduction 26812.2. Determination of Protein Net Charge and Molecular Weight Using

PAGE 26812.3. Determination of Protein Subunit Composition and Subunit Molecular

Weights 270

244

Contents xiii

12.4. Molecular Weight of DNA by Agarose Gel Electrophoresis 27212.5. Identification of Isoenzymes 27312.6. Diagnosis of Genetic (Inherited) Disorders 27412.7. DNA Fingerprinting and Restriction Fragment Length

Polymorphism 27512.8. DNA Sequencing with the Maxam-Gilbert Method 27912.9. Immunoelectrophoresis 282

Suggested Reading 287References 287Problems 288

13. Isoelectric Focusing and 2D Electrophoresis

13.1. Introduction 29013.2. Carrier Ampholytes 29113.3. Modern IEF with Carrier Ampholytes 29313.4. Immobilized pH Gradients (IPGs) 29613.5. Two-Dimensional Electrophoresis 29913.6. Difference Gel Electrophoresis (DIGE) 301

Suggested Reading 303References 303Problems 304

14.Capillary Electrophoresis

14.1. Introduction 30614.2. Electroosmosis 30714.3. Elution of Sample Components 30814.4. Sample Introduction 30914.5. Detectors for Capillary Electrophoresis 310

14.5.1. Laser-Induced Fluorescence Detection 31114.5.2. Mass Spectrometric Detection 31314.5.3. Amperometric Detection 31514.5.4. Radiochemical Detection 318

14.6. Capillary Polyacrylamide Gel Electrophoresis (C-PAGE) 31914.7. Capillary Isoelectric Focusing (CIEF) 321

Suggested Reading 322References 323Problems 323

15.Centrifugation Methods

15.1. Introduction 32515.2. Sedimentation and Relative Centrifugal g Force 325

290

306

325

xiv Contents

15.3. Centrifugal Forces in Different Rotor Types 327

15.3.1. Swinging-Bucket Rotors 32715.3.2. Fixed-Angle Rotors 32815.3.3. Vertical Rotors 328

15.4. Clearing Factor (k) 32915.5. Density Gradients 330

15.5.1. Materials Used to Generate a Gradient 33115.5.2. Constructing Pre-Formed and Self-Generated Gradients 33115.5.3. Redistribution of the Gradient in Fixed-Angle and Vertical

Rotors 333

15.6. Types of Centrifugation Techniques 333

15.6.1. Differential Centrifugation 33415.6.2. Rate-Zonal Centrifugation 33415.6.3. Isopycnic Centrifugation 336

15.7. Harvesting Samples 33615.8. Analytical Ultracentrifugation 336

15.8.1. Instrumentation 33715.8.2. Sedimentation Velocity Analysis 33815.8.3. Sedimentation Equilibrium Analysis 341

15.9. Selected Examples 342

15.9.1. Analytical Ultracentrifugation for Quaternary StructureElucidation 342

15.9.2. Isolation of Retroviruses by Self-Generated Gradients 34315.9.3. Isolation of Lipoproteins from Human Plasma 34415.9.4. Centrifugal Microfluidic Analysis 344

Suggested Reading 346References 346Problems 347

16.Chromatography of Biomolecules 349

16.1. Introduction 34916.2. Units and Definitions 35016.3. Plate Theory of Chromatography 35016.4. Rate Theory of Chromatography 35116.5. Size Exclusion (Gel Filtration) Chromatography 35316.6. Stationary Phases for Size Exclusion Chromatography 358

16.6.1. Particulate Gels 35816.6.2. Monolithic Stationary Phases 360

Contents xv

16.7. Affinity Chromatography 360

16.7.1. Immobilization of Affinity Ligands 36216.7.2. Elution Methods 36416.7.3. Determination of Association Constants by High Performance

Affinity Chromatography 364

16.8. Ion-exchange Chromatography 368

16.8.1. Retention Model for Ion-Exchange Chromatography ofPolyelectrolytes 369

16.8.2. Further Advances in Ion-Exchange Chromatography 374

Suggested Reading 374References 374Problems 375

17.Mass Spectrometry of Biomolecules

17.1. Introduction 37717.2. Basic Description of the Instrumentation 379

17.2.1. Soft Ionization Sources 379

17.2.1.1. Fast Atom/Ion Bombardment (FAB) 38017.2.1.2. Electrospray Ionization (ESI) 38017.2.1.3. Matrix-Assisted Laser Desorption/Ionization

(MALDI) 381

17.2.2. Mass Analyzers 38217.2.3. Detectors 385

17.3. Interpretation of Mass Spectra 38617.4. Biomolecule Molecular Weight Determination 38817.5. Protein Identification 39217.6. Protein-Peptide Sequencing 39317.7. Nucleic Acid Applications 39717.8. Bacterial Mass Spectrometry 39817.9. Mass Spectrometry Imaging 399

Suggested Reading 401References 401Problems 402

18.Micro-TAS, Lab-on-a-Chip, and Microarray Devices

18.1. Introduction 40418.2. Device Fabrication Materials and Methods 40518.3. Microfluidics 405

18.3.1. Fluid Transport 405

377

404

xvi Contents

18.3.2. Valves and Reservoirs 40618.3.3. Mixing and Sample Separation 406

18.4. Detectors 40718.5. Examples of Bioanalytical Devices 407

18.5.1. DNA Separation Using a Nanofence Array MicrofluidicDevice 408

18.5.2. Two Dimensional Electrophoresis on a Microfluidic Chip 40918.5.3. Microfluidic Antibody Capture for Single-Cell Proteomics 41018.5.4. Multiplexed PCR Amplification and DNA Detection on a

Microfluidic Chip 41018.5.5. Silicone Protein Separation Chip Based on a Grafted

Ion-Exchange Polymer 41118.5.6. Circular, Biofunctionalized PEG Microchannels for Cell

Adhesion Studies 411

Suggested Reading 412References 412Problems 413

19.Validation of New Bioanalytical Methods 414

19.1. Introduction 41419.2. Precision and Accuracy 41519.3. Mean and Variance 41619.4. Relative Standard Deviation and Other Precision Estimators 417

19.4.1. Distribution of Errors and Confidence Limits 41819.4.2. Linear Regression and Calibration 41919.4.3. Precision Profiles 42019.4.4. Limit of Quantitiation and Detection 42119.4.5. Linearizing Sigmoidal Curves (Four-Parameter Log-Logit

Model) 42219.4.6. Effective Dose Method 423

19.5. Estimation of Accuracy 424

19.5.1. Standardization 42419.5.2. Matrix Effects 425

19.5.2.1. Recovery 42519.5.2.2. Parallelism 426

19.5.3. Interferences 426

19.6. Qualitative (Screening) Assays 427

19.6.1. Figures of Merit for Qualitative (Screening) Assays 427

Contents xvii

19.7. Examples of Validation Procedures 428

19.7.1. Validation of a Qualitative Antibiotic Susceptibility Test 42819.7.2. Measurement of Plasma Homocysteine by Fluorescence

Polarization Immunoassay (FPIA) Methodology 42919.7.3. Determination of Enzymatic Activity of β-Galactosidase 43319.7.4. Establishment of a Cutoff Value for Semi-Quantitative Assays for

Cannabinoids 434

Suggested Reading 435References 436

Answers to Selected Problems 437

Index 449

Preface to Second Edition

The success of our first edition, with reader feedback, has encouraged us to producean expanded and updated second edition of Bioanalytical Chemistry. Over the pastdecade, we have both read, in the primary literature, about enormous advances incertain areas, including imaging and microfluidic devices. It had also been suggestedthat an introductory chapter involving instrumental measurement principles andmethods would be helpful to readers. New chapters were warranted, and we haveincluded these in our second edition.

Traditional areas of bioanalytical chemistry, such as electrophoresis, chromatog­raphy and immunoassay, have also seen surprising and significant developments. Anenormous development in 2D electrophoresis, involving the simultaneous separationof two or more cell lysates on a single gel, has provided a quantum leap for proteo­mic studies. Nanoparticles, quantum dots and new polymers have seen many appli­cations to biomolecule separation and quantitation. We have attempted to provide anintroduction, as well as leading references, to these areas in our second edition.

The Argentine-Canadian collaboration of the authors began in the mid-1990s asa result of mutual interests in bioanalytical chemistry research and teaching. Over theyears, despite the advantages of modern telecommunications technology, we havefound that travel is both necessary and beneficial, especially when the final stages ofmanuscript preparation are underway. With the north-south travel, we have foundthat the climate is always pleasant somewhere.

SUSAN R. MIKKELSEN

EDUARDO CORTÓN

Buenos Aires, ArgentinaAugust 2015

xix

Preface to First Edition

The expanding role of bioanalytical chemistry in academic and industrial environ­ments has made it important for students in chemistry and biochemistry to be intro­duced to this field during their undergraduate training. Upon introducing abioanalytical chemistry course in 1990, I found that there was no suitable textbookthat incorporated the diverse methods and applications in the depth appropriate to anadvanced undergraduate course. Many specialized books and monographs exists thatcover one or two topics in detail, and some of these are suggested at the end of eachchapter as sources of further information.

This book is intended for use as a textbook by advanced undergraduate chemis­try and biochemistry students, as well as bioanalytical chemistry graduate students.These students will have completed standard introductory analytical chemistry andbiochemistry courses, as well as instrumental analysis. We have assumed familiaritywith basic spectroscopic, electrochemical and chromatographic methods, as theyapply to chemical analysis.

The subject material in each chapter has generally been organized as a progres­sion from basic concepts to applications involving real samples. Mathematicaldescriptions and derivations have been limited to those that are believed essential foran understanding of each method, and are not intended to be comprehensive reviews.Problems given at the end of each chapter are included to allow students to assesstheir understanding of each topic; most of these problems have been used as exami­nation questions by the authors.

As research in industrial, government and academic laboratories moves towardincreasingly interdisciplinary programs, the authors hope that this book will be usedto facilitate, and to prepare students for, collaborative scientific work.

SUSAN R. MIKKELSEN

EDUARDO CORTÓN

Waterloo, CanadaJuly 2003

xxi

Acknowledgments

The support of our colleagues at the University of Waterloo and Universidad deBuenos Aires, during the preparation of this book, is gratefully acknowledged.

We also acknowledge the patience and support of our families and friends, whounderstood the time commitments that were needed for the completion of thisproject.

SUSAN R. MIKKELSEN

EDUARDO CORTÓN

Buenos Aires, Argentina andWaterloo, Canada

xxiii

Chapter 1

Quantitative InstrumentalMeasurements

1.1. INTRODUCTION

This chapter introduces the basic principles underlying many common methods ofsignal transduction. This term is used to describe the conversion of one type ofenergy to another. Generally, analytical specialists use the term transducer todescribe the conversion of a concentration (or mass) into a useful electronic signal,which is ultimately almost always a voltage. This voltage is related to the concentra­tion (or mass) of the analyte, or species of interest, in the original sample. The spe­cies that can be measured by one or more of these methods is not always the analyteitself; for example, if the analyte is an enzyme or other catalytic species, the deple­tion of reactants or accumulation of products is assessed based on their own uniqueproperties.

Transduction can be accomplished in many different ways, and the choice of thebest method depends on which of many possible physical properties are exhibited bythe measured species. In this chapter, we consider the three main types of transduc­tion that are widely used in instrumental methods in bioanalytical chemistry. Theconversion of light into current is performed by photodiodes or photomultipliers,and this current is then electronically converted into a voltage that is proportional tothe intensity of the light. Electrochemical and surface plasmon resonance transducersconvert chemical energy into a measured voltage or into a current that is subse­quently converted to a voltage. Scintillation counters, used in many radiochemicalmethods, first convert beta-particle radioactivity to light, and the light is detectedusing photodiodes or photomultipliers. Thermal transducers, used for calorimetry,convert heat into current (and then voltage).

Considerations for the choice of a transduction method include the uniquenessof the various measurable properties of the measured species, since it is often presentin a complicated sample matrix. The matrix is the surrounding environment, andincludes all other components present in the sample. Matrix components can inter­fere with measurements in direct or indirect ways: a matrix component may exhibit a

Bioanalytical Chemistry, Second Edition. Susan R. Mikkelsen and Eduardo Cortón. 2016 John Wiley & Sons, Inc. Published 2016 by John Wiley & Sons, Inc.

1

2 Chapter 1 Quantitative Instrumental Measurements

similar physical property to the analyte, and interfere with analyte measurement;also, a matrix component may interact with the analyte, changing the nature of itsphysical property and/or the magnitude of its resulting signal.

This chapter is intended as an introduction and brief review of common trans­duction methods used in bioanalytical chemistry. More detailed descriptions ofapplications and instrumental variations will be found within specific chapters ofthis book, where more specialized adaptations are described for specific assaymethods.

The reader is referred to two excellent analytical chemistry textbooks for greaterdepth of coverage of most of the basic descriptions given in this chapter, as well astwo excellent review articles for more information on thermal measurement meth­ods, listed at the end of the chapter.

1.2. OPTICALMEASUREMENTS

The majority of quantitative optical methods make use of light that is either absorbedor emitted in the ultraviolet and visible regions of the electromagnetic spectrum.These regions formally correspond to wavelengths of 1.0× 10-8 to 7.8× 10-7 m, andare more commonly expressed in nm units (10 to 780 nm). The far UV region, alsocalled the vacuum UV region, is generally not analytically useful, but the near UVand the visible regions are widely used.

The colours that surround us result mainly from wavelength-selective visiblelight absorption by molecules present in the items that we see. However, differencesbetween species, and between individuals within a species, cause the wavelengthrange of visible light, and the colours within this range, to be perceived differently.Common examples are bumblebees, that have blue-shifted visible ranges, andhummingbirds, that have red-shifted ranges. For this reason, standard wavelengthranges have been defined for the different colours of the visible spectrum. Forexample, blue light is defined as the 440–470 nm range, and if blue light is absorbed,its complementary colour, orange, is observed. Similarly, if green light(500–520 nm) is absorbed, purple is the observed colour. Many compounds absorblight at multiple wavelengths, and it is the combination of complementary coloursthat we observe.

The relationship between wavelength, frequency and energy of light is shownbelow:

E hυ hc=λ; (1.1)

where E is the energy of the light, h is Planck’s constant (6.626× 10 34 J s), υ is thefrequency of the light (s 1), λ is the wavelength of the light (m), and c is the speed oflight (2.998× 108 m/s in a vacuum, and this number is divided by the refractiveindex n for any other medium). This relationship connects the two key concepts thatlight is both a particle (a photon with energy E) and a wave, with frequency υ andwavelength λ.

31.2 Optical Measurements

In the visible and near UV regions of the spectrum, molecules absorb and emitlight as their electronic configurations change. For example, electrons convertbetween paired and unpaired states, or between bonding and non-/antibonding orbi­tals. These conversions are accompanied by energy gains or losses as the moleculeabsorbs or emits a photon. Depending on molecular structure, as well as many otherfactors including solvent, pH and temperature, fixed electronic energy levels exist,and only photons of particular energies (wavelengths) can be absorbed or emitted.Associated with each electronic energy level are vibrational and rotational energylevels, which are separated by much smaller energy differences. Isolated vibrationalor rotational transitions can be made to occur using infrared or microwave radiation,which have much lower energy. But the electronic transitions that occur in the UV-visible region are accompanied by vibrational and rotational transitions, and thismeans that a range of wavelengths can be absorbed by molecules, shown in Eq. 1.2:

ΔERot; (1.2)ΔET ΔEElec ΔEVib

where, for a given electronic transition, the total energy ΔET of the photons absorbedis the sum of the energy required for the electronic transition itself, ΔEElec, which isfixed, plus the energy changes associated with multiple possible vibrational and rota­tional transitions, ΔEVib and ΔERot. This means that, for any given electronic transi­tion, molecules absorb or emit a fairly wide range of wavelengths, centered on awavelength of maximal absorption or emission. For molecules absorbing or emittinglight in the near UV and visible regions, the range of wavelengths can be as large as100 nm for a given electronic transition, because of these accompanying vibrationaland rotational transitions.

1.2.1. UV-Visible Absorbance

A simple spectrophotometer, an instrument for measuring absorbance, consists of alight source, a monochromator (or filter), a sample compartment and a light detector,all of which are enclosed to prevent interference from ambient light. These compo­nents are shown as a block diagram in Figure 1.1. Typically, the light source is atungsten filament lamp (for the visible region) and/or a deuterium lamp (for the UVregion); both of these sources emit continuous radiation over a wide range of wave­lengths. Wavelength selection can be accomplished using filters, for repetitive fixed-wavelength measurements, or a monochromator containing a diffraction grating orprism, that allows adjustment of wavelength as well as wavelength scanning. Thequality of the filter or monochromator determines the width of the wavelength rangein the light beam that exits the device and is directed into the sample. Analyte solu­tions are contained in a cuvette (or cell) made of a material that is transparent to thewavelength(s) of interest, such as quartz, glass or polystyrene. Light detection maybe accomplished using a photomultiplier tube, a photodiode, or a photodiode array(in which the spatial distribution of light of different wavelengths allows nearlyinstantaneous acquisition of a complete spectrum).

4 Chapter 1 Quantitative Instrumental Measurements

Figure 1.1. Block diagram of a simple UV-Vis absorption spectrophotometer.

Many variations of this simple design have been introduced for specializedapplications. For example, dedicated instruments may employ an inexpensive light-emitting diode as the light source, a combination of absorption and interference fil­ters for wavelength selection, or a flow cell in which a solution continuously flowspast the light beam. In all cases, the instruments are designed to measure the absorp­tion of light by an analyte.

The intensity, or power, of the incident monochromatic beam of light is giventhe symbol PO, and the light intensity that exits the sample compartment has thesymbol P. Commonly, PO is measured using a reagent blank solution, i.e. a solutioncontaining all of the components of the sample solution except the analyte. Transmit­tance, T, is the ratio of these values (P/PO), and may be expressed as this simpleratio, with a value between zero and one, or as a percentage that ranges from zero toone hundred.

As the concentration of the analyte in the sample cell is increased, the transmit­tance decreases, but the dependence of transmittance on concentration is not linear.For quantitative purposes, transmittance values are converted to absorbance (A) val­ues as follows:

A log T log Po=P (1.3)

Absorbance increases linearly as analyte concentration is increased. It alsoincreases linearly with the distance through which the light beam travels in the sam­ple; this is called the path length and is given the symbol b. The Beer-Lambert Law(Eq. 1.4), also called Beer’s Law, is the most important relationship in quantitativespectrophotometry.

A εbc (1.4)

In this relationship, absorbance A depends linearly on analyte concentration cwith two proportionality constants: b, the path length, and ε, the molar absorptivityof the analyte. Absorbance is unitless, and so the units of ε are generally M-1cm-1,when analyte concentration is in molar units and path length is expressed in cm.

Absorbance is additive. If there is more than one absorbing species present in asolution, the total absorbance at a given wavelength is the sum of the absorbances ofthe individual species at that wavelength. This property is analytically useful for thequantitation of multiple absorbing species, if the molar absorptivities are known atmultiple wavelengths. The concentrations of two components, for example, can bedetermined by measuring absorbances at two wavelengths, at which the molarabsorptivities of the two components are known.