Electrokinetic Phenomena: Principles and Applications in Analytical Chemistry and Microchip Technology

473 Pages·2003·9.108 MB·English

by  Rathore, Anurag

Elect ro ki neti c
Phenomena
Principles and Applications in Analytical
Chemistry and Microchip Technology
edited by
Anurag S. Rathore
Amgen, Inc.
Thousand Oaks, California, U.S.A.
Andris Guttman
Diversa Corporation
San Diego, California, U.S.A.
MARCEL
MARCELD EKKERIN, C. NEWY ORK BASEL
DEKKER
Copyright © 2004 Marcel Dekker, Inc.
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Copyright © 2004 Marcel Dekker, Inc.
Foreword
The field of electrokinetic phenomena has had a long history. For over a cen-
tury,ithasbeenrecognizedthationsinsolutionmoveinthepresenceofelectric
fields. It is also well known that bulk flow can occur when electric fields are
appliedandwhenthesurfacescontainingthefluidarecharged(electroosmosis).
These two phenomena, ion and bulk flow movement in the presence of electric
fields, and their applications in both analytical chemistry and microfluidics are
covered in depth in this book.
The book is an outgrowth of the field of electrophoresis, and as such it
owes a debt of gratitude to all the pioneerswho brought the science to where it
is today. While I do not wish to provide a historical survey, it is nevertheless
usefultoexplore brieflythepastso thatthereaderwillunderstand andappreci-
ate the advances outlined in this book.
Electrophoresis, a method of differential migration based on the size-to-
charge ratio of ions in the presence of an applied electric field, had from its
beginningastrongbiologicalfocus.Ittookthisdirectionbecausemanybiologi-
calsubstancesarecharged,aneffectivemeansforsolubilizationinthepredomi-
nantlyaqueousenvironmentofthebiologicalmilieu.TheUppsalaSchool,under
theleadershipofNobelLaureateArneTiselius,enumeratedmanyoftheimpor-
tant first principles of the method before and after World War II. The process
of moving boundary electrophoresis in free solution, for example, was devel-
oped by Tiselius and was widely used for the study of proteins during this era
(1). Later, in order to contain the bulk fluids, electrophoresis was practiced
using support (anticonvective) media, e.g., paper or starch. From these early
choices, gels made of cross-linked polyacrylamide and agarose became widely
used as support media. When the pore sizes of such gels are optimally con-
trolled, the electrophoretic migration of proteins, DNA, and other biopolymers
are retarded as a function of their size, due to interaction with the gel. It was
Copyright © 2004 Marcel Dekker, Inc.
later discovered that denatured proteins in the presence of 0.1% SDS or higher
yielded migration based on molecular weight, as a result of a constant mass-to-
charge ratioof theSDS proteincomplexes. Similarphenomena occurin thegel
separationofsingle-anddouble-strandedDNA,inwhicheachbaseorbasepair
yields a constant increment of mass and charge. Today, separations based on
slab gel electrophoresis are widely practiced, particularly in biological labora-
tories—for example, 2-D gel electrophoresis separation of proteins.
Slabgelelectrophoresishaslimitations,perhapsthemostimportantbeing
that it is difficult to automate. Thus, column or free zone electrophoresis, in
analogy to column liquid chromatography, was a field that early on attracted
attention. Particularly noteworthy was the 1967 publication of the Ph.D. thesis
of Stellan Hjerten, where many of the major principles of the method were
described (2). Unfortunately, in those days relatively wide tubes (3 mm) were
required in order to inject sufficient material to be observed with the detectors
available. It was the development of the appropriate detection and other instru-
mentation that led to capillary electrophoresis (CE) in 1981 by Jim Jorgenson
(3). As demonstrated in that first paper, very high-efficiency separation using
bulk flow electroosmosis was achieved. As discussed in this book, the sharp
band was in large part the result of the plug-like flow of electroosmosis in
contrasttotheparabolicflowprofilegeneratedbypressure-drivenlaminarflow.
The spectacular efficiencies generated by capillary electrophoresis drew
others,suchasmyself,toenterthefield.Fortunately,theHumanGenomeProj-
ect began in 1990, and capillary electrophoresis was one of the technologies to
be developed for DNA sequencing. With linear polymer solutions, enormous
column efficiencies were found, up to 3×107 plates/meter (4). Such perfor-
mance was required to separate consecutive Sanger fragments for which the
mobilitydifferenceswereextremelysmall.Therestishistory,ascapillaryelec-
trophoresis using replaceable polymer matrices in a multiplexed capillary array
format was used to sequence the human genome and is presently being applied
to sequence the genomes of many other organisms.
Capillary electrophoresesalso began thedrive towardminiaturization and
lab-on-a-chip. At its inception was the µTAS concept of Michael Widmer (5).
This lab-on-a-chip method, in which microfluidic devices were manufactured
using a methold similar to that for semiconductor devices, first focused on the
transfer of CE methods from the capillary to the chip. Today, electrophoresis
utilizingelectricfieldsforbulkflow,injection,gradientmixing,andotherappli-
cations,iswelladvanced,asdescribedinthisbook.Inaddition,otherinnovative
approaches to the use of electric fields have been introduced. For example,
capillary electrochromatography (CEC), in which electric fields are employed
to drive fluids across chromatographic packed beds, has been advanced and is
also discussed in this book.
Copyright © 2004 Marcel Dekker, Inc.
Electrokinetic phenomena is no longer merely a laboratory curiosity. It is
akeycomponentofpresent-daymicroscaleanalysis.Lookingtowardthefuture,
it may well be that some of the current techniques will be supplemented by
nanotechnologyapproaches,butfluidcontrolandionmovementwilllikelystill
involve electric fields. This book will afford the reader an appreciation of the
power of electrokinetic phenomena. I congratulate the editors on the breadth of
coverage and the timeliness of the topic.
Barry L. Karger
Barnett Institute
Northeastern University
Boston, Massachusetts, U.S.A.
REFERENCES
1. TiseliusA.Anewapparatusforelectrophoreticanalysisofcolloidalmixtures,Trans
FaradaySoc1937;33:524.
2. HjertenS.Freezoneelectrophoresis.ChromatogrRev1967;9:122–219.
3. Jorgenson JW, Lucas KD. High-performance separations based on electrophoresis
andelectroosmosis.JChromotogr1981;218:209–216.
4. GuttmanA,CohenAS,HeigerDNandKargerBL.Analyticalandmicropreparative
ultrahigh resolution of oligonucleotides by polyacrylamide gel high-performance
capillaryelectrophoresis.AnalChem1990;62:137–141.
5. ManzA,GraberN,andWidmerHM.Sens.ActuatorsB1990;1:244–248.
Copyright © 2004 Marcel Dekker, Inc.
Preface
Electrokineticphenomenarepresentthebasisofdifferentelectric-field-mediated
separation techniques in use today. These analytical tools, including capillary
zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC),
capillary electrochromatography (CEC), capillary gel electrophoresis (CGE),
andotherformsofcapillaryelectrophoresis,aregainingmoreandmorepopular-
ity in the analytical labs of pharmaceutical and biotechnology companies as
orthogonaltechniquestotraditionalchromatographiclybasedmethodsofanaly-
sis. The recent great interest in microfabricated separation devices has led to
newer challenges in the areas of system design, as well as application of the
various electrokinetic phenomena for optimized and efficient separations. The
goal of this book is to address both the various underlying fundamentals in this
area and the numerous applications of these electro-driven separation tools. In
thisspirit,thebookincludescontributionsfromactiveresearchersinbothacade-
mia and industry to present a complete picture of where this field is at present
and where and how it is evolving.
Thefirstchapterdiscussesthevariouselectrokineticinteractionsthatgov-
ernmigrationandseparationofdifferentsamplecomponentsintwoofthemost
commonly used electrodriven analytical techniques—CZE and CEC—in order
to provide a platform of fundamental understanding for more detailed and spe-
cific chapters that follow.
The next four chapters discuss the basic principles underlying operation
and method development of the most common electrodriven analytical tech-
niques: CE, capillary isoelectric focusing (cIEF), capillary gel electrophoresis
(CGE), and affinity capillary electrophoresis (ACE). Weinberger presents a
comprehensive approach for method development in CE with an emphasis on
small-molecule applications. This is followed by Kila´r’s chapter describing the
principles of and method development in cIEF, as well as recent innovations
Copyright © 2004 Marcel Dekker, Inc.
and applications. Guttman’s chapter covers the theoretical and practical aspects
of capillarygel electrophoresisand reviewsthe keyapplication areasof nucleic
acid, protein and complex carbohydrate analysis, affinity-based methodologies,
and related microseparation methods such as ultra-thin-layer gel electrophoresis
andelectric-field-mediated separationsonmicrochips. OkunandKenndlerreview
two approaches to performing ACE—the equilibrium case for the study of weak
tomoderate affinities and the nonequilibrium caseformoderate tohigh affinities.
The practicaladvantagesand limitations of bothmodesofACEare discussed.
The next three chapters discuss the various fundamental and practical as-
pects of separations via CEC. Wen et al. review the theoretical basis of the
generationandcontrolofelectroosmoticflowinCEC,followedbyadiscussion
of various factors that can influence the separation speed, including novel col-
umndesigns.BartleandMyersreviewtheprogressmadetowardunderstanding
some of the factors that influence the performance of silica-based columns in
CEC. Various practical aspects of CEC are addressed, including column pack-
ing, frit formation, and column repeatability and reproducibility. The impact of
propertiesofstationary andmobilephasesonCECseparation isalsodiscussed.
Stol and Kok present a novel method for measuring electroosmotic pore flow
inpackedCECcolumns.Theoreticalandexperimentalresultsarepresentedand
used as the basis for guidelines for optimizing the stationary-phase pore size
and the ionic strength of the mobile phase.
The next four chapters discuss the extension of electric-field-mediated
separationtoolstothenano-world.Tsuda’schapterdescribesdesignandperfor-
manceofanultra-shortcolumnCECsystem.Columnpreparationanddesignof
the pumping system, injector, and detector are discussed. Laurell et al. discuss
theimpactofmicrostructuredevelopmentsontheneweraofproteomics.Issues
associated with design of a lab-on-a-chip system are discussed, and it is shown
that microfluidic modeling and simulation by in silico technology are highly
useful in the first attempts at hypothesis testing. Kikutani and Kitamori report
recent developments in the fields of microunit operations (MUOs), continuous-
flow chemical processing (CFCP), and thermal lens microscopy (TLM) detec-
tion for microchips. The application of these methodologies to integrate a vari-
etyofchemicalandanalyticalprocessesonamonolithicmicrochipisexamined,
and their use for highly sensitive wet analysis of heavy metal ions, diagnostic
immunoassays, and rapid and high-yield chemical synthesis is discussed.
Khandurina’schapterpresentsasurveyofthemostrecentadvancesinCE-based
preparative methods with special attention to microscale fraction collection and
on-line sample preparation. These chapters suggest that emerging novel micro-
fabricated devices will further increase the level of integration and multifunc-
tionality in chemical and biochemical processing technology.
The last three chapters discuss some of the most important applications
of the techniques mentioned above. Sweedler and coworkers describe a novel
Copyright © 2004 Marcel Dekker, Inc.
combinationofCEandCECwithnuclearmagneticresonance (NMR)spectros-
copy (CE-NMR and CEC-NMR). The chapter presents the theory and instru-
mentationofthishyphenatedtechnique, alsoexaminingissuesregardingdesign
oftheinterfaceandfinally,applications.RemchoreviewsvariousCECapplica-
tions, including analysis of inorganic anions and cations, pollutants, amino
acids, peptides, proteins, carbohydrates, nucleotides and their derivatives, phar-
maceuticals, and different chiral compounds. Landers’ group reviews clinical
uses of microfluidic devices. These include analysis of clinically relevant ana-
lytessuchassmallmolecules(drugs,ions,neurotransmitters,carbohydrates,and
amino acids), proteins and peptides, and nucleic acids. Further, challenges of
integrating the individual processes into a single device are explored.
Thebookisintendedtoprovideausefulsummaryofthevariouselectroki-
neticphenomenathatplayafundamentalroleinthevariousmethodsandappli-
cations mentionedabove. Inaddition toserving asa referencebook foranalyti-
cal chemists in pharmaceutical and biotechnology companies, the material
should be helpful to graduate students in many disciplines including chemistry,
pharmacy, biochemistry, chemical engineering, and microfluidics.
Anurag S. Rathore
Andra´s Guttman
Copyright © 2004 Marcel Dekker, Inc.
Contents
Foreword
Preface vii
Contributors xiii
1.MigrationofSampleComponentsinCapillaryAnalytical
Techniques:Chromatography,Electrophoresis,
andElectrochromatography1
Anurag S. Rathore
2.MethodsDevelopmentforCapillaryElectrophoresiswith
EmphasisonSmallMolecules15
Robert Weinberger
3.CapillaryIsoelectricFocusing43
Ferenc Kila´r
4.CapillaryGelElectrophoresisandRelated
MicroseparationTechniques69
Andra´s Guttman
5.AffinityCapillaryElectrophoresis109
Vadim M. Okun and Ernst Kenndler
6.ElectroosmoticMobilityandConductivityinMicrochannels141
Emily Wen, Anurag S. Rathore, and Csaba Horva´th
xi
Copyright © 2004 Marcel Dekker, Inc.
7.FactorsInfluencingPerformanceinCapillary
ElectrochromatographyonSilicaColumns167
Keith D. Bartle and Peter Myers
8.EffectsofPoreFlowonSeparationEfficiencyinCapillary
ElectrochromatographywithPorousParticles189
Remco Stol and Wim Th. Kok
9.Ultrashort-ColumnCapillaryElectrochromatography211
Takao Tsuda
10.MicrostructureandIn-SilicoDevelopmentsforHigh-Sensitivity
ProteomicsResearch223
Thomas Laurell, Johan Nilsson, and Gyo¨rgy Marko-Varga
11.MicroChemicalProcessingonMicrochips253
Yoshikuni Kikutani and Takehiko Kitamori
12.MicropreparativeApplicationsandOn-LineSampleTreatment277
Julia Khandurina
13.NMRDetectioninCapillaryElectrophoresisandCapillary
Electrochromatography311
Dimuthu A. Jayawickrama, Andrew M. Wolters,
and Jonathan V. Sweedler
14.ApplicationsofCapillaryElectrochromatography345
V. T. Remcho, StaceyL . Clark, AngelaDoneau ,
and Gabriela S. Chirica
15.ClinicalApplicationsofMicrofluidicDevices427
Joan M. Bienvenue, James Karlinsey, James P. Landers,
and Jerome P. Ferrance
Copyright © 2004 Marcel Dekker, Inc.