Capillary Electrophoresis and Microchip Capillary Electrophoresis Principles, Applications, and Limitations

Explores the benefits and limitations of the latest capillary electrophoresis techniques

Capillary electrophoresis and microchip capillary electrophoresis are powerful analytical tools that are particularly suited for separating and analyzing biomolecules. In comparison with traditional analytical techniques, capillary electrophoresis and microchip capillary electrophoresis offer the benefits of speed, small sample and solvent consumption, low cost, and the possibility of miniaturization.

With contributions from a team of leading analytical scientists, Capillary Electrophoresis and Microchip Capillary Electrophoresis explains how researchers can take full advantage of all the latest techniques, emphasizing applications in which capillary electrophoresis has proven superiority over other analytical approaches. The authors not only explore the benefits of each technique, but also the limitations, enabling readers to choose the most appropriate technique to analyze a particular sample.

The book's twenty-one chapters explore fundamental aspects of electrophoretically driven separations, instrumentation, sampling techniques, separation modes, detection systems, optimization strategies for method development, and applications. Specific topics include:

• Critical evaluation of the use of surfactants in capillary electrophoresis
• Sampling and quantitative analysis in capillary electrophoresis
• Capillary electrophoresis with electrochemical detection
• Overcoming challenges in using microchip electrophoresis for extended monitoring applications
• Capillary electrophoresis of intact unfractionated heparin and related impurities
• Microchip capillary electrophoresis for in situ planetary exploration

Each chapter begins with an introduction and ends with conclusions as well as references to the primary literature. Novices to the field will find this book an easy-to-follow introduction to core capillary electrophoresis techniques and methods. More experienced investigators can turn to the book for troubleshooting tips and expert advice to guide them through the most advanced applications.

Carlos D. García, PhD, is an Associate Professor of Analytical Chemistry at the University of Texas at San Antonio, USA. His group is currently focused on the development of novel bioanalytical strategies involving microfluidics and nanomaterials.

Karin Y. Chumbimuni-Torres, PhD, is a Research Associate at the University of Texas at San Antonio, USA. She is interested in pursuing the development of electrochemical biosensors and their integration to microchip-based platforms.

Emanuel Carrilho, PhD, is an Associate Professor at the University of Säo Paulo, Brazil. With more than twenty-five years of experience in separation science, his group is focused on the development of analytical methods and instrumentation for bioanalyses.

Chapter 1: Critical Evaluation of the Use of Surfactants in Capillary Electrophoresis
Jessica Felhofer, Karin Chumbimuni-Torres, Maria F. Mora, Gabrielle Haby, and Carlos D. García

1. Introduction

2. Surfactant Structures and Properties

3. Surfactants for Wall Coatings

3.1. Controlling the Electroosmotic Flow

3.2. Preventing Adsorption to the Capillary

4. Surfactants as Buffer Additives

4.1. Micellar Electrokinetic Chromatography

4.2. Microemulsion Electrokinetic Chromatography

4.3. Non-Aqueous Capillary Electrophoresis with Added Surfactants

5. Surfactants for Analyte Preconcentration

5.1. Sweeping

5.2. Transient Trapping

5.3. Analyte Focusing by Micelle Collapse

5.4. Micelle to Solvent Stacking

5.5. Combinations of Preconcentration Methods

5.6. Cloud Point Extraction

6. Surfactants and Detection in CE

6.1. Mass Spectrometry

6.2. Electrochemical Detection

7. Conclusions

8. References

Chapter 2: Sample Stacking - A Versatile Approach for Analyte Enrichment in CE and Microchip-CE
Bruno Perlattia, Emanuel Carrilhob,*, Fernando Armani Aguiarc

1. Introduction

2. Isotachophoresis

3. Chromatography based sample stacking

4. Methods based on electrophoretic mobility and velocity manipulation (Electrophoretic Methods)

4.1. Field Enhanced Sample Stacking (FESS)

4.2. Field Enhanced Sample Injection (FESI)

4.3. Large Volume Sample Stacking (LVSS)

4.4. Dynamic pH Junction

5. Sample Stacking in pseudo-stationary phases

5.1. Field Enhanced Sample Stacking

5.2. Hydrodynamic injection techniques

5.2.1. Normal stacking mode (NSM)

5.2.2. Reversed electrode polarity stacking mode (REPSM)

5.2.3. Stacking with reverse migrating micelles (SRMM)

5.2.4. Stacking using reverse migrating micelles and a water plug (SRW)

5.2.5. High-conductivity sample stacking (HCSS)

5.3.E lectrokinetic injection techniques

5.3.1. Field-enhanced sample injection (FESI-MEKC)

5.3.2. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM)

5.4. Sweeping

5.5. Combined techniques

5.5.1. Dynamic pH Junction-Sweeping

5.5.2. Selective Exhaustive Injection (SEI)

5.6. New Techniques

6. Stacking techniques in Microchips

7. Concluding Remarks

8. References

Chapter 3: Sampling and Quantitative Analysis in Capillary Electrophoresis
Petr Kubáo*, Andrus Seiman, Mihkel Kaljurand

1. Introduction

2. Injection techniques in CE

2.1 Hydrodynamic sample injection

2.1.1 Principle

2.1.2 Advantages and performance

2.1.3 Disadvantages

2.2  Electrokinetic sample injection

2.2.1 Principle

2.2.2 Advantages and performance

2.2.3 Disadvantages

2.3 Bias free electrokinetic injection

2.4 Extraneous sample introduction accompanying injections in CE

2.5 Sample stacking

2.5.1 Principle

2.5.2 Advantages and performance

2.5.3 Disadvantages

2.6 Alternative batch sample injection techniques

2.6.1 Rotary type injectors for CE

2.6.2 Hydrodynamic sample splitting as injection method for CE

2.6.3 Electrokinetic sample splitting as injection method for CE

2.6.4 Dual-opposite end injection in CE

3. Micromachined/microchip injection devices

3.1 Droplet sampler based on digital microfluidics

3.2 Wire loop injection

4. Automated flow sample injection and hyphenated systems

4.1 Introduction

4.2 Advantages and performance

4.3 Disadvantages

5. Computerized sampling and data analysis

6. Sampling in portable CE instrumentation

7. Quantitative analysis in CE

7.1 Introduction

7.2 Quantitative analysis with HD injection

7.3 Quantitative analysis with EK injection

7.4 Validation of the developed CE method

7.5 Computer data treatment in quantitative analysis

8. Conclusions

9. References

Chapter 4: Practical considerations for the design and implementation of High Voltage Power Supplies for Capillary and Microchip Capillary Electrophoresis
Lucas Blanes1, Wendell Karlos Tomazelli Coltro2,3, Renata Mayumi Saito4, Claudimir Lucio do Lago3,4, Claude Roux1 and Philip Doble1.

1. INTRODUCTION

1.1 - High Voltage Fundamentals

1.2 - Electroosmotic flow control

1.3 - Technical aspects

1.4 - Construction of Bipolar HVPS from unipolar HVPS

1.5 - Safety considerations

1.6 - HVPS commercially available

1.7 - Practical considerations

1.8 - Alternative sources of HV

1.9- HVPS controllers for MCE

2- High Voltage measurement

3. Concluding Remarks

4. References

Chapter 5: ARTIFICIAL NEURAL NETWORKS IN CAPILLARY ELECTROPHORESIS
Josef Havel, Eladia María Peña-Méndez, Alberto Rojas-Hernández

1. Introduction

2. Optimization in CE. From single variable approach towards Artificial Neural Networks

2.1 Limitations of “traditional” single variable approach

2.2 Multivariate approach with Experimental design and response surface modeling

2.2.1 Experimental design

2.2.2 Response surface modeling

3. Artificial Neural Networks in electromigration methods

3.1 Introduction - basic principles of ANN

3.2 Optimization using a combination of ED and ANN

3.2.1 Testing of ED-ANN algorithm

3.2.2 Practical applications of ED-ANN

3.3 Quantitative CE analysis and determination from overlapped peaks

3.3.1 Evaluation of calibration plots in CE using ANN to increase precision of analysis

3.3.2 ANN in quantitative CE analysis from overlapped peaks

3.4 ANN in CEC and MEKC

3.5 ANN for peptides modeling

3.6 Classification and fingerprinting

3.7 Other applications

4. Conclusions

5. Acknowledgements

6. References

Chapter 6: IMPROVING THE SEPARATION IN MICROCHIP ELECTROPHORESIS BY SURFACE MODIFICATION
M. Teresa Fernández-Abedul1, Isabel Álvarez-Martos1, Francisco Javier García Alonso2, Agustín Costa-García

1. Introduction

2 Strategies for improving separation

2.1 Selection of an adequate technique: ME

2.2 Microchannel design

2.3 Selection of an appropriate ME material

2.4 Optimization of the working conditions

2.5 Surface modification

2.5.1 Surface micro and nanostructuration

2.5.2 Employment of energy sources

2.5.3 Chemical surface modification

2.5.3.1 Application to bioassays

2.5.3.2 When the surface modification is made?

2.5.3.3  Where does the modification takes place?

2.5.3.4 How is the modification performed?

2.5.3.5  What electrophoresis mode is the modification made for?

3 Chemical modifiers

3.1  Surfactants

3.2 Ionic liquids

3.3 Nanoparticles

3.4 Polymers

4. Conclusions

5. Acknowledgements

6. References

Chapter 7: Capillary Electrophoretic Reactor and Microchip Capillary Electrophoretic Reactor: Dissociation Kinetic Analysis Method for “Complexes” Using Capillary Electrophoretic Separation Process
Toru Takahashi and Nobuhiko Iki

1.Introduction

2.Basic concept of CER

3. Dissociation kinetic analysis of metal complexes by CER

3.1 Derivation of the Rate Constants of Dissociation of 1:2 complexesof Al3+ and Ga3+ with an azo dye ligand, 2,2’-dihydroxyazobenzene- 5,5’-disulfonate in CER.

3.2 Expanding the scope of the CER to measurement of fast dissociation kinetics with a half-life from seconds to dozens of seconds: Dissociation kinetic analysis of metal complexes by microchip capillary electrophoretic reactor (μCER)

3.3 Expanding the scope of the CER to measurement of slow dissociation kinetics with a half-life of hours

4. Principle of LS-CER

4.1 LS-CER of Ti(IV)-Catechin Complex

4.2 LS-CER of Ti(IV)-Tiron Complex.

5. Expanding the scope of the CER to measurement of dissociation kinetics of biomolecular complexes

6. Dissociation kinetic analysis of [SSB-ssDNA] by CER

7. Conclusions

8. References and Notes

Chapter 8: Capacitively Coupled Contactless Conductivity Detection (C4D) Applied to Capillary Electrophoresis (CE) and Microchip Electrophoresis (MCE)
José Alberto Fracassi da Silva, Claudimir Lucio do Lago, Dosil Pereira de Jesus, Wendell Karlos Tomazelli Coltro

1. Introduction

2. Theory of C4D

2.1 Basic principles of C4D

2.2 Simulation

2.3 Basic Equation for Sensitivity

2.4 Equivalent Circuit of a CE-C4D System

2.5 Practical Guidelines

3. C4D Applied to Capillary Electrophoresis

3.1 Instrumental Aspects in CE

3.2 Coupling C4D with UV-Vis Photometric Detectors in CE

3.3 Fundamental Studies in Capillary Electrophoresis using C4D

3.4 Fundamental Studies on C4D

3.5 Applications

4. C4D Applied to Microchip Capillary Electrophoresis

4.1 Geometry of the detection electrodes

4.1.1 Embedded electrodes

4.1.2 Attached electrodes

4.1.3 External electrodes

4.2 Applications

4.2.1 Bioanalytical Applications

4.2.2 On-chip enzymatic reactions

4.2.3 Food Analysis

4.2.4 Explosives and Chemical Warfare Agents

4.2.5 Other Applications

5. Concluding Remarks

6. Acknowledgements

7. References

Chapter 9: Capillary Electrophoresis with Electrochemical Detection
Blánaid White

1.Introduction

2. Principles of electrochemical detection

2.1 Amperometric Detection

2.2 Potentiometric detection

2.3 Conductivity Detection

3. Interfacing amperometric detection to capillary electrophoresis

6.1 Off-column detection

6.2 End-column detection

6.2.1 Use of multiple detection electrodes

6.2.2 Pulsed Amperometric Detection

4. Non-aqueous EC detection

5. Electrode material

6. Dual Conductivity and Amperometric Detection

7. Interfacing electrochemical detection to microfluidic capillary electrophoresis

9.1 End-column detection

9.1.1 Pulsed amperometric detection

9.2 Off-channel detection

9.3 Electrode material

8. Portable CE and MCE systems

9. Applications of CE/MCE with AD

10. Future directions for CE/MCE with EC detection

11. References

Chapter 10: Overcoming Challenges in using Microchip Electrophoresis for Extended Monitoring Applications
Scott D. Noblitt and Charles S. Henry

1. Introduction

2.Background Electrolyte (BGE) Longevity

3. Achieving Rapid Sequential Injections

4. Robust Quantitation

5. Conclusions

6. References

Chapter 11: Distinction of coexisting protein conformations by capillary electrophoresis
Hanno Stutz

1. Introduction

2. Protein misfolding and induction of unfolding

3. Conformational pathologies

4. Distinction between Conformations

5. Relevance of conformations for biotechnological products

6. Conformational elucidation – an overview of alternative methods to CE

7. HPLC in conformational distinction

7.1 Intact proteins

7.1.1 Reversed-Phase (RP)-HPLC

7.1.2 Size exclusion (SEC)-HPLC

7.1.3 Ion-exchange-HPLC

7.2 HPLC with detectors sensitive for conformations and aggregates

7.3 Peptides as model compounds for hydrophobic stationary phases in HPLC

8. Capillary electrophoresis (CE) in conformational separations

8.1 Fundamental aspects and survey of pitfalls

8.2 Electrophoretic mobility of proteins

8.3 Peak profiles and derivable thermodynamic aspects of protein re-/unfolding

8.4 Dipeptides as a case study for isomerization

8.5 Denaturation factors and strategies applied in CE

8.5.1 Separation electrolyte, injection solution and sample storage

8.5.2 Denaturation by urea, dithiotreitol and GdmCl

8.5.3 Effects of pH and organic solvents

8.5.4 Temperature

8.5.5 Electrical Field

8.5.6 Detergents

8.5.7 Ligands and ions – case studies on potential amyloidogenic β2m

8.5.8 β-amyloid peptides

8.5.9 Prions

9. Comparison between CE with HPLC

10. Conclusive discussion and method evaluation

10.1 General aspects

10.2 HPLC

10.3 CE

11. References

Chapter 12: Capillary electromigration techniques for the analysis of drugs and metabolites in biological matrices: a critical appraisal
Cristiane Masetto de Gaitani, Anderson Rodrigo Moraes de Oliveira, and Pierina Sueli Bonato

1. Introduction

2. Strategies to obtain reliable capillary electromigration methods for the bioanalysis of drugs and metabolites

2.1 Selectivity and Detectability

2.2 Repeatability

3. Selected applications of capillary electromigration techniques in bioanalysis

3.1 Pharmacokinetics and metabolism studies

3.2 Enantioselective analysis of drugs and metabolites

3.3 Biopharmaceuticals  or biotechnology-derived pharmaceuticals

3.4 Therapeutic drug monitoring

3.5 Clinical and forensic toxicology

4. Concluding remarks

5. References

Chapter 13: Capillary electrophoresis and multi-color fluorescent DNA analysis in an optofluidic chip
Chaitanya Dongre, Hugo J.W.M. Hoekstra, and Markus Pollnau

1. Introduction

2. Optofluidic integration in an electro-phoretic microchip

2.1 Sample fabrication

2.2 Optofluidic characterization

3. Fluorescence monitoring of on-chip DNA separation

3.1 Experimental materials and methods

3.2 Experimental results and analysis

4. Toward ultrasensitive fluorescence detection

4.1 Optimization of the experimental setup

4.2 All-numerical post-processed noise filtering

5. Multi-color fluorescent DNA analysis

5.1 Dual-point, dual-wavelength fluorescence monitoring

5.2 Modulation-frequency encoded multi-wavelength fluorescence sensing

5.3 Application to multiplex ligation-dependent probe amplification

6. Conclusions and outlook

7. Acknowledgements

8. References

Chapter 14: Capillary Electrophoresis of Intact Unfractionated Heparin and Related Impurities
Robert Weinberger

1. Introduction

2. Capillary electrophoresis and heparin

3. Method Development in Capillary Electrophoresis

4. Common Impurities Found in Heparin

5. The United States Pharmacoepia and CE of Heparin

6. Interlaboratory Collaborative Study

7. Conclusions

8. References

Chapter 15: Microchip Capillary Electrophoresis for in situ Planetary Exploration
Peter A. Willis and Amanda M. Stockton

1. Introduction

2. Instrument Design

3. Instrumentation External to the Microdevice

4. Microdevice Basics

4.1 All-Glass Devices for Microfabricated Capillary Electrophoresis

4.2 Three-Layer Hybrid Substrate Glass-Pdms Devices for Fluidic Manipulation

4.3 Integrating Fluidic Manipulation with Electrophoresis

5. Microdevices and their Applications

5.1 Microdevices With Bus-Valve Control Of Microfluidic Manipulation

5.2 Automaton Devices for Programmable Microfluidic Manipulation

6. Conclusions

7. Acknowledgments

8. References

Chapter 16: Rapid Analysis of Charge Heterogeneity of Monoclonal Antibodies by Capillary Zone Electrophoresis and Imaged Capillary Isoelectric Focusing
Yan He, Jim Mo, Xiaoping He and Margaret Ruesch

1. Introduction

2. Capillary Zone Electrophoresis

2.1 Separation and Detection Strategy

2.1.1 Capillary construction

2.1.2 Buffer composition

2.1.3 Separation Voltage and Field Strength

2.1.4 Detection

2.2 Applications

3. Imaged capillary isoelectric focusing

3.1 Method Development and Optimization

3.1.1 Carrier Ampholyte

3.1.2 Additives

3.1.3 Focusing time and voltage  

3.1.4 Salt concentration

3.1.5 Protein concentration

3.2 iCE Method Validation

3.3 Applications 

3.3.1 Cell line development support

3.3.2 Formulation screening

3.3.3 Characterization of acidic species

4.Summary

5. References

Chapter 17: Application of capillary electrophoresis for high-throughput screening of drug metabolism
Roman ?emínek, Jochen Pauwels, Xu Wang, Jos Hoogmartens, Zdenik Glatz, Ann Van Schepdael

1. Introduction

2. Sample deproteinization

3. On-line pre-concentration

4. Method Development

4.1 Dynamic coating of inner capillary wall

4.2 Short-end injection

4.3 Strong rinsing procedure

4.4 Optimized method

5. Method validation

6.Method’s applications

6.1 Drug stability screening

6.2 Kinetic study

7.Conclusion

8.Acknowledgement

9. References

Chapter 18: Electrokinetic Transport of Microparticles in Microfluidic Enclosure Domain
Qian Liang, Chun Yang, and Jianmin Miao

1. Introduction

2. Numerical Model

2.1 Problem description

2.2 Mathematical model

3. Numerical Simulation

4. Results and Discussion

4.1 Particle transport in the bulk flow

4.1.1 The particle velocity in the confined domain

4.1.2 The trajectory of particle transport within the confined domain

4.1.3 The effect of side wall zeta potential on the particle motion

4.2 Particle transport near the bottom surface

4.2.1 The effect of the EDL thickness on the near wall motion of the particle

4.2.2 The effect of surface charge on the near wall transport of the particle

5. Model Application

6. Conclusions

7. References

Chapter 19: Integration of nanomaterials in capillary and microchips electrophoresis as a flexible tool
Germán A. Messina, Roberto A. Olsina, Patricia W. Stege

1. Introduction

1.1 Historical overview of nanotechnology

1.2 Nanomaterials

1.2.1 Carbon-based nanomaterials

1.2.2 Metal-based nanomaterials

1.2.3 Dendrimers

1.2.4 Composites

2. Nanomaterial in Analytical Chemistry

3. Nanoparticles in Capillary electrophoresis

3.1.1 Nanoparticles in capillary electrochromatography

3.1.1.1 Organic Nanoparticles

3.1.1.2 Inorganic particles

3.1.2 Nanoparticles in electrokinetic chromatography

3.1.2.1 Organic nanoparticles

3.1.2.2 Inorganic particles

3.1.3 Nanoparticles in microchip electrochromatography

4. Conclusions

5. References

Chapter 20: Microchip Capillary Electrophoresis to Study the Binding of Ligands to Teicoplanin Derivatized on Magnetic Beads
Toni Ann Riveros, Roger Lo, Xiaojun Liu, Marisol Salgado, Hector Carmona and Frank A. Gomez

1. Introduction

2. Experimental Section

2.1 Materials and Methods

2.2 Procedures

3. Results and Discussion

3.1 FAMCE Studies

3.2 MFAC Studies

4. Conclusions

5. Acknowledgments

6. References

Chapter 21: Glycomic Profiling through Capillary Electrophoresis and Microchip Capillary Electrophoresis
Yehia Mechref

1. Introduction

1.2 Release of N-Glycans from Glycoproteins

1.2.1 Chemical Release

1.2.2 Enzymatic Release

1.3 Release of O-Glycans from Glycoproteins

1.3.1 Chemical Release

1.3.2 Enzymatic Release

2. General Considerations of Capillary Electrophoresis and Microchip Capillary Electrophoresis of Glycans

4.1 Capillary Electrophoresis- Laser-Induced Fluorescence (CE-LIF) Analysis of Glycans

4.2 Interfacing Capillary Electrophoresis and Capillary Electrochromatography to Mass Spectrometry

4.2.1 ESI Interfaces for Capillary Electrophoresis

4.2.2 Sheathless-flow Interface

4.2.3 Sheath-flow Interface

4.2.4 Liquid Junction Interface

4.2.5 MALDI Interfaces for Capillary Electrophoresis

4.2.6 CE-MS Analysis of Glycans

4.2.7 Glycomic Analysis by CEC-MS

3. Microchip Capillary Electrophoresis

4. Conclusions

5. References

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