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. Althoughgreatcarehasbeentakentoprovideaccurateandcurrentinformation,neither theauthor(s)northepublisher,noranyoneelseassociatedwiththispublication,shallbe liableforanyloss,damage,orliabilitydirectlyorindirectlycausedorallegedtobe causedbythisbook.Thematerialcontainedhereinisnotintendedtoprovidespecific adviceorrecommendationsforanyspecificsituation. Trademarknotice:Productorcorporatenamesmaybetrademarksorregisteredtrade- marksandareusedonlyforidentificationandexplanationwithoutintenttoinfringe. LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress. ISBN:0-8247-4306-7 Thisbookisprintedonacid-freepaper. 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Currentprinting(lastdigit): 10 9 8 7 6 5 4 3 2 1 PRINTEDINTHEUNITEDSTATESOFAMERICA 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.