聚合物陶瓷复合材料

Field-FlowFractionation:AddressingtheNanoChallenge

S.KimRatanathanawongsWilliams,J.RayRunyon,andAkramA.AshamesColoradoSchoolofMines

Field-flowfractionationiscomingofageasafamilyofanalyticalmethodsforseparatingandcharacterizingmacromolecules,nanoparticles,andparticulates.Thecapabilitiesandversatilityofthesetechniquesaredis-cussedinlightofthechallengesthatarebeingaddressedinanalyzingnanometer-sizedsamplecomponentsandtheinsightsgainedthroughtheiruseinapplicationsrangingfrommaterialssciencetobiology.(Tolistentoapodcastaboutthisfeature,pleasegototheAnalyticalChemistrymultimediapageatpubs.acs.org/page/ancham/audio/index.html.)In1966,thelateJ.CalvinGiddingsintroducedfield-flowfraction-ation(FFF),achromatography-liketechniquewithanessentiallyone-phasenaturethatgivesitapotentialadvantageinseparatingmacromoleculesandcolloids.1Theensuingyearsbroughttre-mendousinnovationsininstrumentationthatutilizeddifferenttypesoffields,advancesinunderstandingthebehavioroftheselargeanalytesinlaminarflowstreamsinconfinedchannels,andnumerousapplicationsthatdemonstratedtheversatilityofFFF.However,asrecentlyasthemid-1990s,FFFwasstilldescribedasaninterestingtechniquelookingforaniche.Researchinfieldssuchasnanoscienceandnanotechnologyandbiotechnologysubsequentlyexploded.Itisrecognizedthatthesenanometer-sizedanalytesareusuallynotmonodispersedandinfactcanundergodynamicinteractionsthatcausequantitativeshiftsinsubpopulations.Newmetrologiesandanalyticalmethodstoinvestigatepropertiesandvalidatetheoreticalandbehavioralmodelsinthissizeregimewereurgentlyneeded.Asthe“nano”revolutionunfolds,challengesarisefromthediverseprimaryandsecondarypropertiessuchassize,shape,architecture,composition,opticalandelectronicproperties,andtoxicityofnanoparticles,polymers,andnanocomposites.Regula-toryagenciesareconsideringmorerigorousmonitoringofaggregatesinprotein-basedpharmaceuticalsandvaccinesbecauseofconcernsaboutreducedefficaciesandpotentialimmunogenicresponses.Manyquestionsaboutthefunctionofsubcellularcomponentsthatwerepreviouslydisregardedhavebecomethefocusofattention;someofthesecomponentsarealsopotential10.1021/ac101759zXXXXAmericanChemicalSociety

ROBERTGATESdiseasebiomarkers.Separatingthesecomplexandpolydispersesamplesintosimplerunitsforfurtheranalysisandtestingiskeyingainingdetailedinsightsthatwouldotherwisebeunachievable.FFFisafamilyoftechniquesthatwasdesignedtoseparateandmeasurephysicochemicalpropertiesofcomplexmacromo-lecular,colloidal,andparticulatematerials.1-3Theseparationoccursbydifferentialdisplacementinaflowingstreamofliquidthatcarriestheseparatedcomponentstoadetector.Physico-chemicalpropertiesofthespeciesdeterminethemeasuredretentiontime,tr,andthuscharacterizationisusuallyachievedsimultaneouslywithseparation.Theeasyelutionandcollectionofhighlyuniformsamplecomponentsfromthechanneloutlet

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A

isamajorstrengthofFFFtechniques.Eachfractioncanbeexaminedeitherusingofflinetechniquesoronlinemethodssuchaslightscattering,MS,andelementspecificdetectorstoobtaincomplementaryinformationaboutdifferenttypesofdistributions.TheseparationmechanismandopenparallelplateFFFchanneldesignresultsinotherimportantcharacteristicssuchaslowshearrates(idealforinvestigatingfragilespecies),lowsampleloss,tunableselectivityandspeed,applicabilitytodiversesamplesspanningabroadmolecularweight/sizerange,andabilitytoseparateaccordingtodifferentphysicochemicalproperties.

ThegoalsofthisarticlearetobrieflyrevisitthebasicprinciplesofFFF;demonstratethestrengthsoftheFFFfamilyoftechniquesandhowtheyhavebeenusedtoaddressanalyticalchallengesencounteredwithnanometer-sizedanalytespeciesandcomplexsamples;highlightrecentadvancesinFFFinstrumentationinclud-ingtheimportanceofhyphenatedmethods;andincreasetheawarenessofresearcherswhomaybenefitfromFFF’smanycapabilitiesforanalyzingmacromoleculesandnanoparticles.(“Nano”iscommonlyusedinreferencetomatterwithdimensions∼1-100nmthatexhibituniquephenomena.4Theauthorshaveincludedmacromoleculesinthisdiscussionbecausetheyareofteninthenanometersizerange.)BASICPRINCIPLES

FFFseparationisanessentialstepinmeetingthenanochallengebecausemanysamplespossesscontinuousdistributionsinoneormorepropertiesand/orconsistofmultiplecomponentsthatspanawidesizeormolecularweightrange.FFFtakesplaceinathin,ribbon-likechannelinwhichalaminarflowwithaparabolicflowvelocityprofileissubjectedtoaperpendicularlyappliedexternalfield.2TheunderlyingprincipleofallFFFtechniquesisbasedonthebalancebetweenfield-inducedmasstransportofanalytetowardsanaccumulationwallanddiffusionawayfromthiswallcausedbytheresultingconcentrationgradient.Thisleadstotheformationofsamplecloudswhich,atsteadystate,haveequilibriummeanlayerthicknessesl1,l2,...etc.withdifferentlvaluescorrespondingtodifferentflowvelocitystreamlinesoftheparabolicflowprofileandthusdifferenttr.Ideally,eachsamplecomponenthasauniquelorD/|U|whereDisthediffusioncoefficientandUisthefield-inducedmigration.ThesmallertheDand/orthelargertheinteractionwiththefield,thesmallerthelvalueandthelongerthesamplecomponentremainsintheFFFchannel.Therelationshipbetweenl,tr,andtheforce,F,exertedonasingleparticleormacromoleculebytheappliedfieldcanbeapproximatedbytheequation2,3tr)

w|F|wt

0

6l)6kT

(1)

wheret0isthevoidtime,wisthechannelthickness,kis

Boltzmann’sconstant,andTisabsolutetemperature.Theassumptionsarethatallparticlesarenoninteractingpointmassesandw.l(necessaryforefficientseparation).TheaboveequationshowsthatFgovernsbothretentionandseparationbecausedifferencesintheforceexperiencedbyvariousparticles,∆F,willresultinroughlyproportionaldifferencesintr.ThemagnitudeofFand∆Fdependsonparticleproperties,fieldstrength,andthetypeoffieldemployed.

B

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Virtuallyanytypeoffieldcanbeusedaslongasitinteractswithsomephysicochemicalpropertyofthesamplecomponentstodrivethemtotheaccumulationwall.ThishasgivenrisetomultipletechniqueswithintheFFFfamily,manyofwhichhaveyettobecommercialized,e.g.,dielectrophoretic,magnetic,electri-cal,andacoustic.5-8Thermal,sedimentation,andcrossflowFFFarecommerciallyavailableandarethusthemostcommonlyusedFFFtechniques.ThermalFFF(ThFFF).ThisfirstexperimentallyrealizedFFFtechniqueutilizesatemperaturegradienttodrivesamplestotheaccumulationwall.9Theribbon-likechannelissandwichedbe-tweentwoheatconductiveblocksthatareindividuallytemperaturecontrolled,onewithheatingelementsandtheotherwithcirculat-ingchilledwaterorcoolant.Temperaturedifferencesbetweenthehotandcoldwallscanbeashighas∼100K,whichwhenappliedacrossthechannelthicknessof∼10-2cm,translatestogradientsthatare∼104Kcm-1.ThFFFwasoriginallyusedforsyntheticpolymersinorganicsolventsbuthasbeenexpandedtonanoparticlesinaqueousandnonaqueoussolvents.10,11TheeffectivedrivingforceoneachpolymermoleculeisF)kTDTdTDdx

(2)

whereDTisthethermaldiffusioncoefficientanddT/dxistheappliedtemperaturegradient.CombiningEquations1and2revealsthattrisproportionaltothefieldstrength(dT/dx)andtheSoretcoefficient,DT/D,andthatthemeasurementoftryieldsDT/D.IfDTisknown,Dcanbecalculated,whichsubsequentlyyieldsmolecularweightMWbecauseofthedependenceD=A(MW)-bwhereAisanexperimentallydeterminedproportionalityconstantandbis∼0.6forathermodynamicallyfavorablesolvent.Alternatively,ifDisknown,DTcanbecalculated.ThFFFisoneofthemostintriguingtechniquesoftheFFFfamilybecausetheDTtermcanbeusedtoaddressanalyticalchallengesassociatedwithpolymerandnanoparticleinterfacialcompositionandpolymermicrostructureandarchitecture.12However,retentiontimesaredifficulttocalculatebecausethermaldiffusioninliquidsisnotfullyunderstoodandDTvaluesspecifictotheparticularpolymerandnanoparticlearenotwidelyavailable.ThissituationisbeingremediedbycouplingThFFFtodetectorsthatmeasureMWindependentlyandbyongoingeffortstoevaluateexistingtheoriesforpredictingDT.13SedimentationFFF(SdFFF).TheFFFchannelencirclesaspinningcentrifugebasketthatgeneratesdifferentialaccelerationforcesondifferentparticles.14Theforceexperiencedbyaparticlecanbewritteninvariousformstoshowtherelationshipbetweenthefieldstrength(oracceleration,G)andtheeffectivemass(truemassminusbuoyantmass),m′,particlevolume,Vp,diameter,d,andthedifferenceindensitybetweentheparticleandcarrierliquid,∆F.

F)m󰀁G)Vp|∆F|G)

π6

d3

|∆F|G(3)

SubstitutingFintoEquation1yieldsaproportionalitybetweentrandd3thatexplainsSdFFF’sabilitytobaselineresolveparticleaggregatesrangingfromsingletstooctuplets.15A10×differ-

enceindwillyielda1000×differenceintroradiameter-basedselectivity(definedas|dlogtr/dlogd|)of3.Thoughthislevelofselectivityishighlydesirableincertainsituations,analysesforpolydispersesamplesarelengthy.Thisisreadilyresolvedbydecreasingthefieldstrengthaccordingtoatheoreticalfunctionthatmaintainsaconstantfractionatingpowerthrough-outtheseparation.3Theproportionalitybetweentrandm′isalsoimportantbecauseitallowsSdFFFtobeusedasamicrobalanceformeasuringminute(10-16-10-18g)changesinnanoparticleeffectivemass.16,17CrossflowFFF(FlFFF).CrossfloworflowFFFisthemostcommonlyusedFFFtechnique.Acrossflowoffluidtransportsthesampletotheaccumulationwallwhich,inthiscase,isasemipermeablemembranelaidoverasupportingfritpanel.18TheoreticalcalculationshaveconfirmedthatthetypicalFlFFFcrossflowflowratesdonotperturbtheparabolicflowprofile.19Intheory,FlFFFisuniversallyapplicabletoallanalytespecies.Inpractice,thelowersizelimitissetbythemolecularweight-orsize-cutoffofthemembrane,whichdeterminesnotonlytheanalytespeciesretainedinthechannel,butalsothehighestcrossflowratesthatcanbeused.Thedrivingforceexertedonasingleparticleisrelatedtothefrictioncoefficient,f,viscosity,η,andtheStokesorhydrodynamicdiameter,d,bythefollowingseriesofequations:F)f|U|)kT

|U|

D

)3πη|U|d(4)

whereUissynonymouswithcrossflowvelocity.Thisequation(andEquation1)showsthattrisdependentontheanalyte’sdiffusioncoefficient,whichinturnisrelatedtoasphereequivalenthydrodynamicd.Assumingallparticlesinthesamplearesimilarinshape,qualitativeinterpretationofthemeasuredfractogramisstraightforwardbecausetrispropor-tionaltodandthefractogramreflectsthesizedistribution.DifferentvariantsoftheFlFFFchanneldesignincludetheoriginalsymmetricchannelwithtwopermeablewalls,today’smostcommonlyusedasymmetricchannelwithonenon-permeablewall(AsFlFFF,alsoknownasAF4),20andthehollowfiberchannel.21UnlikeThFFFandSdFFFchannels,whichareconstructedfromruggedmetallicmaterials,therelativelydelicatemembraneaccumulationwallinFlFFFchannelsrequirescarefultreatmentandperiodicreplacement.

DielectrophoreticFFF(DEP-FFF).Dielectrophoresisisthemigrationofaparticleinanon-uniformelectricfieldcausedbyelectrostaticinteractionsbetweenthefield-inducedpolarizationintheparticleandthefield.5,22TheDEPforceexperiencedbyasphericalparticleofradiusrisrelatedtothepermittivity(ordielectricconstant)ofthemedium,εs,andtheimposedmogeneouselectricfield,binho-E

.F)2πr3εsfcmb∆

|Eb|2(5)

TheClausius-Mossottifactor,fcm,takesintoaccountcomplexpermittivitiesoftheparticleandthesuspendingmediumanddeterminestherelativestrengthanddirectionoftheDEPforceonaparticleasafunctionoftheappliedfieldfrequency.Hence,particlescanbeseparatedinaDEP-FFFsystemonthebasis

ofdifferencesineffectivedielectricproperties,suchassemi-conductorparticlebandgapsandelectroniccharacteristics.23DEPforcesalsodependonthespatialdistributionoftheappliedelectricalfield,andadvancesinmicrofabricationtechnologieshaveenabledtheproductionofinterdigitatedmicroelectrodearrayscapableofproducinglargeDEPforceswithsmallappliedvoltages.5,22MagneticFFF(MgFFF).VariousMgFFFinstrumentcon-figurationshaveachievedvaryingdegreesofsuccess.ThemostrecentisaquadrupoleMgFFF(QMgFFF)instrumentcomprisedofahelicalseparationchannelmountedinanaxisymmetricmagneticfield.6Theoutwardlyradialmagneticfieldsetsupafieldgradientacrossthechannel,andthemagnitudeofthegradient’sinteractionwiththemagneticcomponentoftheparticlesdeter-minestr.Theforceexperiencedbyasingleparticleundertheinfluenceofthemagneticfieldgradientisgivenby

F)VmM|∇B|

(6)

whereVmisthevolumeofthemagnetizedcomponentofthe

particle,Misthemagnetizationofthiscomponent(whichisrelatedtomagneticsusceptibility),and∇BisthegradientinmagneticfieldB.

Summary.Equations1-6describethenormalmodesepara-tionmechanismthatanalytessmallerthan∼1µmexperienceandexplainwhysmall(highdiffusioncoefficient)analyteselutefirst.Foranalytes>∼1µm,thesteric-hyperlayerseparationmechanismreverseselutionorder,3butthisFeaturefocusesonnano-sizedmaterials,whichareusuallyseparatedbythenormalmode.Thesesixequationsalsoprovideinsightintoexperimentalparametersandsamplepropertiesthatgovernretention,thepropertiesbywhichthesamplecomponentscanbesortedintoaseriesofmoreuniformbins,andtheinformationthatcanbegarneredusingeachtypeoffield.Moreover,theelutionprofileorfractogramcanbetransformedintodistributionsspecifictothefieldthatwasemployed,e.g.,sizedistributionsforFlFFFormassordensitydistributionsforSdFFF.Increasingly,FFFcoupledwithorthogonalmethodsprovidescomplementaryinformationandthusamorecompletepictureofsamplecharacteristics.ThisnextsectiondemonstratestheimportantfeaturesofFFF,drawingonexamplesthatincludemacromoleculesandnanoparticles.FFFSEPARATIONANDANALYSIS

SelectivityandComplexSamples.FFFtechniquesseparateanalytesaccordingtodifferentpropertiesdependingonthefieldapplied.Theselectivityoftheseparationdependsontherelation-shipbetweenretentiontimeandthespecificsamplepropertybeingprobed.Forexample,becausetr∝d3forSdFFFandtr∝dforFlFFF,thediameter-basedselectivitiesare3and1,respectively.Thehighertheselectivity,thesmallerthediffer-enceinsizethatcanbedifferentiatedbyretentiontime.Thisisthefigureofmeritintheseparationofpolydispersesamplesbecausethecontinuousdistributionsinsize,mass,etc.areexpectedtoproducebroadelutionprofiles.Differentcompo-nentseluteatdifferenttimeseventhoughdistinctpeaksarenotevident.Hence,thetraditionalmetricofnumberoftheoreticalplatesisnotasusefulfordescribingtheeffective-nessoftheseparationofnon-uniformnano-sizedmaterials.

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Figure1.AsFlFFFanalysisofribosomalsamplestakenin(a)theexponentialbacterialgrowthphaseand(b)5hafterinoculationinthemidexponentialphase.Thetwotracesin(b)representabsorbanceatλ)260nm(A)and280nm(B).Reprintedfromref24withpermissionfromElsevier.

Themainchallengesinbiotechnologyareunderstandinggrowthpropertiesofcellculturesandoptimizingproteinyields.Forexample,thetotalamountofribosomalmaterialpercellandtherelativeamountoftheassembled70Sribosomeareindicatorsofthecells’proteintranslationcapacity.Thus,separationandquantitationoftypesofribosomalmaterialcanprovideinvaluableinsightsforestablishingoptimumbioreactorconditions.Figure1demonstratesthelevelofresolutionobtainedbyAsFlFFFinthelownanometersizeregioninacomplexribosomalsample.24TheAsFlFFFfractogramsshowthatthe30Sand50Sribosomalsubunits(∼16and18nm)and70Sassembledunit(∼25nm)areclearlyresolvedandseparatedfromtRNAandproteinswithin8min.Theanalysistimewasoptimizedsothatasamplecanbewithdrawnfromthebioreactor,processed,andanalyzedin16mincycles.TheadvantageofaseparationstepoversimplymeasuringtheaveragesizeofcomponentsintheentiresampleisclearlydemonstratedbyFigure1b.Thefractogramfromasamplecollectedatthemid-exponentialgrowthphaseshowsapeakthatisbelievedtobe100Sribosomalparticles,acomponentthatwouldhavebeenmissedifonlytheaveragediameterwasmeasured.Similarseparationsareobtainablefornanoparticles.25WideMolecularWeightandSizeRange.Samplesareroutinelyfilteredpriortochromatographicseparations,andthecolumnsthemselveshavebuilt-infritstoremovelargesamplecomponentsthatcanbedeleterioustocolumnperformance.BecauseFFFchannelsareopenstructureswithtypicalthick-nessesof∼100-500µm,oftentimestheentiresamplecanbeanalyzedwithoutfiltration.Figure2showsThFFFfractogramsforfilteredandunfilteredpoly(vinylacetate)(PVAc)samplesandD

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Figure2.ThFFF-MALSanalysisofultrahighMWPVAc.(a)MALSfractogramandMWsand(b)dRIfractogramandrrmsofunfiltered(red)andfiltered(blue)PVAc.(I90:lightscatteringintensityat90°;Vr:retentionvolume).Reprintedfromref26withpermissionfromElsevier.

their(a)MWand(b)rootmeansquareradius,rrms,obtainedbyonlinemultianglelightscattering-differentialrefractiveindex(MALS-dRI)detection.26ThisdetectorcombinationconfirmsthesuccessofaseparationthatspansfiveordersofmagnitudeinMWandatrrmsvaluesupto400nm.ThelowshearratesencounteredinFFFchannelssignificantlyreducethepossibil-ityforsheardegradationofultrahighMWpolymers.ThedRIplotinFigure2brevealsthatfiltrationunexpectedlyremovesasignificantamountoflowerMWsolublepolymers,whichleadstoerroneousresults.Thisstudydemonstratedthattheroutinesamplefiltrationusedasastandardstepinmanymacromolecularanalysesmustbeperformedjudiciously.SizeSeparation.Sizeandsizedistributionsplayimportantrolesinopticalandelectricalproperties,fateandtransport,andbiologicalefficacy.FFFhasbeenusedtocharacterizeawidevarietyofnanoscalematerials,includingengineerednanoparticles(quantumdots,25,27,28fullerenes,29andcarbonnanotubes30);environmentalcolloids;31-33naturalandsyntheticmacromole-cules;26,34-37proteins,proteinaggregates,andproteinvirus-likeparticles;38-44drugandgenetherapydeliveryvehicles;45-47andsubcellularparticles.24Nanoparticleshapehasdramaticeffectsonopticalproperties.Forexample,goldnanorods(GNRs)havebeenproposedforapplicationssuchasbioimagingandbiosensingbecausedifferentaspectratiosoftherodswillhavecharacteristicabsorbancewavelengthsthatcanbediscernedinamultiplexedanalysis.TheuseofFFFtoachievewhatiseffectivelyashape-basedseparationofapolydisperseGNRsampleisshowninFigure3.FractionsF1-F4werecollectedattheindicatedtimeintervalsinFigure3aandcontainedGNRsubpopulationsofincreasinglengthasevidencedbytransmissionelectronmicroscopy(TEM)imagesFigure3.(a)AsFlFFFfractogramofGNRs.Thecross-hatchedregionsdesignateretentiontimesoffourdifferentcollectedfractions.(b)TEMimagesandassociatedsolutioncolorsofcollectedfractions.

inFigure3b.AsuspensionofeachfractionisshownbelowitscorrespondingTEMimage;thedifferentcolorsfurtherconfirmthepresenceofdifferentGNRsubpopulations.48ThisGNRseparationisbasedonthesphereequivalenthydrodynamicdiameterasdescribedbyEquation4.Thoughthisisnotatrueshapeseparation,thisexampledemonstratesthepossibilityofobtainingfractionscontainingdifferentaspectrationanoparticles.FlFFF-MALShasalsobeensuccessfullyusedtoseparateandcharacterizeDNAstabilizedsinglewallednanotubesinanaqueouscarrierliquidaccordingtolength.30Mass.Functionalizednanoparticlesareusedinmanyapplica-tionsrangingfromimmunodiagnosticstoreinforcedplastics.Theabilitytoquantitatetheamountof(physicallyorchemically)attachedfunctionalitiescanprovideinsightsintothebehaviorandperformanceofthesenanoparticles.SdFFFiswellsuitedforthistaskbecausetrisproportionaltom′oftheparticle(Equation3).Hence,theadsorptionorattachmentofproteins,polymers,orotherfunctionalitiestothesurfaceofnanoparticlesisreflectedbyanincreaseintr.SdFFFhasbeenusedasamicrobalanceformeasuringminute(10-16-10-18g)changesinm′andmonitoringdifferentstagesinthedevelopmentofananopar-ticle-basedbioluminescentimmunosensor.17Thisbiosensor,whichhasadetectionlimitof15fmol,consistedofpolystyrene(PS)latexparticlesthatwerefirstcoatedwithasurfactantandthenwithbovineserumalbumin(BSA).SdFFFmeasurementsyieldedasaturationlevelloadingof700nmolpyruvatekinase-IgY(antiBSA)conjugateper7.5×1010BSAcoatedparticlesandanaverageof22BSAmoleculesperpyruvatekinase-IgYconjugate.

Composition.Elasticity,adhesiveness,haziness,andphaseseparationbehaviorareexamplesofpolymerpropertiesthatFigure4.Composition-basedThFFFseparations.(a)PSandPBApolymerswithsimilarhydrodynamicradii.(b)Core-shellnanoparticleswithdifferentPBA:PMAAshellcompositions.Latex1:135nmPScore;Latex2:9%PMAAshell;Latex3:15%PMAAshell;Latex4:36%PMAAshell.Partbreprintedfromref10withpermissionfromElsevier.

dependoncomposition.ThFFFcanseparateorganosolublemacromoleculesbycomposition,despiteanincompleteunder-standingofthermaldiffusion.Figure4ashowsabaselineresolvedcompositionseparationofPSandpoly(n-butylacrylate)(PBA)polymerswithsimilarhydrodynamicradii,rh,(12.75nm)andD(4.0×10-7cm2sec-1).ThFFFhasalsobeenusedtoseparatenanoparticleswithdifferentsurfacecompositions.Figure4bshowsthesuperimposedfractogramsoffourcore-shelllatexparticlesconsistingofa135nmPScoreandshellswithvaryingratiosofPBAandpoly(methacrylicacid)(PMAA).10ThenanoparticlewiththelargestshellratioofPBA/PMAAhadtheshortestretentiontime,whereasthePScore(smallestparticle)wasretainedlongest,suggestingasurfacecompositiondependentretention.DTisauniquepropertyofthepolymer-solventpairandvarieslinearlywithcopolymercomposition.Therefore,ThFFFcandeterminethecopolymercompositionofanunknownsamplefrommeasuredtrandD.49Thesamehasbeendemonstratedforthecore-shellnanoparticlesmentionedabove.10BandGap.Carbonnanotubeshavebeenproposedforap-plicationssuchashighstrengthandconductivecomposites,sensors,fieldemissiondisplays,andhydrogenstoragemedia.Theirsemiconductiveandopticalpropertiesdependontheirchiralityandphysicaldimensions.30Dielectrophoreticforceshavebeenusedtoseparatesinglewallnanotubes(SWNTs)accordingtoelectronictypeandtoproducefractionsthatareenrichedindifferentSWNTdiameters.23DifferencesindielectricconstantsformthebasisfortheseDEP-FFFseparations.Becausethedielectricconstantisgovernedbythebandgapwhich,inturn,isinverselyproportionaltodiameter,separationsbasedonbandgapsanddiameterarepossible.TheformerwasachievedbyutilizingAnalyticalChemistry,Vol.xxx,No.xx,MonthXX,XXXX

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Figure5.AsFlFFFfractionationandcharacterizationofprotease-resistantprionproteinparticles(PrPres)withonlineMALS,dRI,andQELS.(a)Specificinfectivityofeachfractionoverlaidwiththecorrespondingmolarmass(i.e.,molecularweight,MW),rrms(i.e.,radiusofgyration),andhydrodynamicradius(rh).(b)TEMimagesoffourfractionsshowinganincreaseinsizeandfibrillengthcorrespondingtoincreasingfractionnumbers(scalebars)100nm).Reprintedfromref40withpermissionfromNaturePublishingGroup.

electricfieldconditionsthatpreferentiallytrappedmetallicspeciesontheelectrodewhilethesemiconductorspeciesflowedthroughtheDEP-FFFchannel.Inthelattercase,theenrichmentofsemiconductorSWNTsofdifferentdiameterswasachievedbytheusualFFFseparationmechanism.Thatis,differencesindielectrophoreticforcescausedSWNTstoresideindifferentflowvelocitystreamlinesintheDEP-FFFchannelandeluteatdifferenttimes.FluorescenceandRamanspectrawerecollectedassup-portingevidenceofseparationandenrichment.MagneticSusceptibility.Thelargesurfaceareaofmagneticnanoparticlesmakethemattractivecandidatesforapplicationssuchasdrugtargeting,catalysis,andbioseparation.6Thesynthesisofmonodispersemagneticnanoparticlesisdifficulttoachieve,yetrequiredformedicalapplications.Forinstance,tominimizetheunwantedsystemiceffectsofpharmaceuticalmagneticnanopar-ticlesandallowtheuseofhigherandeffectivedrugdoses,theweaklymagneticfractionmustbedepletedfromthepolydisperseformulation.Therewasnomethodtomeasurethepolydispersityinthemagneticpropertiesofthesepharmaceuticalnanocarriersuntilrecently,whenQMgFFFsuccessfullydifferentiatedthreedifferentlotsofmagneticnanoparticles6andfractionatedpolydis-persesamplesofdextran-coatedmagnetitenanoparticlesaccord-ingtotheirmagneticsusceptibility.COMPLEXSAMPLESANDPROCESSES

Highselectivity,widesizerange,andlowshearratesmakeFFFideallysuitedfortheseparationofcomplexandoftenfragilesamples.Biologicalandenvironmentalsamplestendtobehighlycomplexwithmultiplecomponentscomposedofwidearraysofphysicochemicalpropertiesthatcanundergodynamicchanges.(TheribosomedatashowninFigure1isagoodexampleofacomplexsamplewhosecomponentscanshiftwithgrowthcondi-tionsandtime.)Thus,itisnotsurprisingthatFFF(particularly,FlFFF)hasfoundanicheintheseareas.38-47ProteinAggregation.Misfoldedandpartiallyunfoldedpro-teinscanleadtotheformationofaggregatesanddepositssuchasthefibrilsandplaquesassociatedwithneurodegenerativediseases.Proteinaggregatescausedbychemical-orphysical-inducedinstabilitiesduringmanufacturing,storage,and/orad-ministrationcanalsobepresentinbiotherapeuticproducts.Thelinkbetweenproteinaggregatesandimmunogenicity,thedifficultyofestablishingcause-and-effect,andtheneedforanappropriatesuiteofinstrumentationforanalyzinge10µmaggregatesarecurrentlytopicsofdiscussioninthisfield.50-52KeychallengestoF

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thecharacterizationofproteinaggregatesinproteintherapeuticsincludequantitationandidentificationofaggregateswithawidesizerangeof0.1-10µm.AnoptimizedAsFlFFF-MALS-UVmethodwasdevelopedthatcoveredthe50-500nmsizerange.39Proteinself-associationattributedtotheAsFlFFFsamplefocusingstepwasavoidedbyusingmoderateflowrates.Quantifyingminorspecies,e.g.,highorderaggregates,remainsanunresolvedproblembutcouldbeaddressedwithmoresensitivedetectors.Astudyofatherapeuticimmunoglobulinfurtherillustratesthecomplexityofaggregate-containingsamples.38AhostofanalyticalmethodsincludingAsFlFFFandfluorescencemicroscopyshowedthatdifferenttypesofaggregatescanformthroughnoncovalentbonds(e.g.,hydrogenbonding,electrostaticrepulsionandattrac-tion,andhydrophobicinteractions).TheflexibilityinthechoiceofFFFcarrierliquidwasanadvantagebecauseaminoacid-orformulation-basedbufferscouldbeused.Pathologically,proteinaggregationisassociatedwithdiseasessuchasAlzheimer’s,Parkinson’s,andtransmissiblespongiformencephalopathies.Inagroundbreakingstudy,AsFlFFFwithMALS-dRI-quasielasticlightscattering(QELS)detectionwasessentialindemonstratingthataggregatesoftheprotease-resistantprionprotein(PrPres)withMWof300-600kDa,hydrodynamicradiusrhof14-28nm,andthatarecomposedof14-28PrPresmoleculespossessedthegreatestinfectivity(Figure5a).40TEManalysisoffourfractionscollectedacrossthefractogramshowedanincreaseinbothsizeandfibrilformationconcurrentwithincreasingAsFlFFFelutiontimes(Figure5b).ThechangeintherhtorrmsratioasdetectedbyMALSandQELScorroboratedthepresenceoffibrilsinthelonger-retainedfractions.Theseresultssupportanemergingviewthatwithproteinaggregationdiseases,smallersubfibrillarparticlesmaybemuchmorepathologicalthanlargeramyloidfibrilsorplaques,whichhasimplicationsonthedevelopmentoftherapiestotreatthesediseases.40DrugandGeneDelivery.Sizeandsizedistributionsareamongtheimportantparametersthatgoverndrugandgenedeliveryefficiencies.AsFlFFFcoupledwithlightscatteringdetec-torsisacharacterizationmethodforliposomepreparations53andbiodegradableN,O-dimethacryloylhydroxylamine(DMHA)cross-linkedpoly(N-isopropylmethacrylamide)(PNiPAM)thermosen-sitivenanogelsthatarebeingdevelopedforuseasdrugdeliveryvehicles.45DMHAdecomposesunderphysiologicconditionsandhaslowinvivotoxicity.Thesize,molarmass,topology,particlenumberdensity,andbyproductsfromtemperatureandpHdependenterosionstudiesweremonitored.Atphysiologicalconditions,thenanogel’sweightaveragemolecularweightde-creasedfrom6×107to2×107gmol-1withoutasignificantincreaseinsize.Acomparisonofrhtorrmsrevealedthatthenanogeltopologyremainedconstantdespiteextensiveerosion,whichwasattributedtotheself-cross-linkingbehaviorofthePNiPAM.Theseobservationssubstantiatedanefficientdesignofananogelscaffoldingfordrugrelease.

FlFFF-MALShasmonitoredthesizeandstabilityofcationicsurfactant-basednon-viralvectorsforgenetherapy.46TheadditionofUV-visandQELSdetectorstoAsFlFFF-MALSenabledthesimultaneousmeasurementofcompositionandamountoffreepolycationanddetectionofaggregatesinthefinalformulationofchitosan-basedvectors,whichsubsequentlyallowedcorrelationwithcelltransfectionrates.47Thedevelopmentofagenedeliveryvehiclebasedonvirus-likeparticlesderivedfromthehumanpolyomaJC-virusagainreinforcedthenecessityofusingasuiteofdetectorstoobtainmultiplelevelsofinformation.41Theseanalysesprovidedinsightsintoproceduresandformulationsessentialforoptimalstabilityandtransfectionratesandcouldnothavebeenachievedwithoutaseparationstage.NanoparticleToxicity.Thepotentialrisksofnanomaterialstohumanhealthandtheenvironmenthavedrawnagreatdealofattentioninthelastseveralyears.TheNationalNanotechnologyInitiativeheldworkshopsandsubsequentlypublisheddocumentsthatidentifypriorityareasforenvironmental,health,andsafetyresearchneedsforengineerednanoscalematerials.4Onekeygoalistheestablishmentofmethodsfornanoparticlequantificationandcharacterization.Thisisparticularlyimportantbecausethephysicochemicalpropertiesofnanomaterialsvarysubstantiallyfrombatch-to-batchandvendor-to-vendor,whichhasledtoinconsistentperformanceandbehaviorcharacteristics.FlFFFwithinductivelycoupledplasmaMS(ICPMS)wasusedtostudyshorttermexposuretoxicityof2and5nmCdSe/ZnSquantumdots(QD)withtwodifferentsurfacecoatings,11-mercaptoundecanoicacid(MUA)andpolyethyleneoxide(PEO).27ThemorestablePEO-QDwerelessacutelytoxictoDaphniamagnathantheMUA-QD,whichisdesignedforaccelerateddissolution.DissolvedCd2+waspresentintheMUA-QDsuspen-sion,andthustoxicitywasattributedtocadmiumpoisoning.ThePEO-QDsuspensioncontainednodissolvedCd2+,whichindicatedadifferenttoxicitymechanism.Theresultsofthisstudysuggestedthatasmallcoresizedoesnotnecessarilytranslatetogreatertoxicityandthattheuseofparticlenumberormassasadosemetriccanleadtodifferentconclusions.INSTRUMENTALADVANCES

DevelopmentsinFFFinstrumentationinthelastdecadehaveaddressednonidealbehaviororextendedFFFcapabilities.ThecouplingofFFFwithMALS-dRI,QELS,andMShasbeenbeneficialtoeachtechnique.ThetrainofmoremonodispersesampleselutingfromtheFFFchannelalleviatestheproblemofselectivedetectionandbiastowardscomponentsthatproducethestrongestresponses.54Ontheotherhand,thesedetectorscompensatefornonidealbehavior,e.g.,analyte-accumulationwallinteractionsthataffectFFFretentiontimes.OrthogonaltechniquessuchasICPMS,LC,andESIMSprovideawealthofinformationandhaveopenedgatewaystonanoparticletoxicityandproteomicstudies.27,44SomeadvancesinFFFinstrumentationandap-proachesarediscussedbelow.OnlineConcentration.Dilutionisacommonproblemforelution-basedseparationmethods.FFFseparationtakesplaceintheregionneartheaccumulationwall,sothemajorityoftheFFFchannelisoccupiedbyfluidonly.Removaloffluidfromtheregionfurthestfromtheaccumulationwallresultsinsampleconcentra-tion.ThishasbeenaccomplishedbyintroducingaslotoutletpositionedontheoppositechannelsurfacetotheaccumulationwallatthedetectorendoftheFFFchannel.55Carrierliquidiscontinuouslyremovedthroughtheslotoutletleadingtoon-linesampleconcentrationjustpriortodetection.A14×increaseinsampleconcentrationhasbeendemonstrated.55,56Anotherusefulapproachfortheanalysisofdilutesamplesistheopposedflowsampleconcentrationmethod.57AlargevolumeofsampleisintroducedintotheFlFFFchannel,whereitencoun-tersanopposingflow.TheratiooftheopposingflowratetosampleintroductionflowratedeterminesthepointalongtheFFFchannelwheresampleisfocusedpriortothestartoftheseparation.Becauseonlyparticle-freefluidcanexitthroughthesemipermeablemem-braneaccumulationwall,thesampleisconcentratedonthemem-brane.Samplevolumesaslargeas1Lhavebeenloadedintoa∼1.3mLvolumeFlFFFchannel,resultinginconcentrationfactorsashighas105.However,dependingontheflowratesused,sampleloadingcanbetimeconsuming.Thisapproachwasdemon-stratedinasymmetricFlFFFchannelbutcanalsobeimplementedinotherFlFFFconfigurations.

HighThroughputSeparation.Parallelseparationscanin-creasesamplethroughput.ThishasbeenachievedbyarrangingmultipleFlFFFchannelsincircularorparallelconfigurations.44,58,59Offlinecombinationof2Disoelectricfocusing(IEF)-AsFlFFF44andmultiplexedhollowfiber(MxHF5)-FlFFF59withnanoflowLCESIMS/MSallowedprofilingofurinaryproteinsandphospho-lipidsinlipoproteins.IEF-AsFlFFFcharacterizedhumanurinaryproteinsinlessthan30minutes.AnIEFseparationoftheproteinsbyisoelectricpointwasfollowedbysixmultiplexedsize-basedAsFlFFFfractionations,eachatauniquepH.Shotgunproteomicsfromeachsamplefractionidentified245totaland110novelproteins.TheMxHF5-FlFFFexperimentscombinedsixhollowfiberchannelsinparallelforthesemi-preparativesortingandcollectionoflipoproteins.Thehollowfiberchannelisdisposable,whichreducescross-contaminationrisksofclinicalsamples.Microchannels.MiniaturizedchannelshavebeenintroducedformanyFFFsystemssuchasThFFF,AsFlFFF,andelectricalFFF7,44,60Thereductioninchanneldimensionsoffersadvanta-geousfeaturessuchaslowsampleconsumptionandshorteranalysistimesatlowerenergyandresourcecosts.HighTemperatureandNonaqueousFlFFF.Ahightem-peratureFlFFFinstrumenthasbeenrecentlyintroducedfornonaqueousseparations.Newlydevelopedceramicmembranesstableat130°CandcompatiblewithchlorinatedorganicsolventsformtheheartofthestainlesssteelchannelthatwasusedtofractionatesemicrystallineultrahighMWpolyethylene(PE).35,36High-temperatureAsFlFFFcombinedwithonlineIR,MALS,andviscometrydetectionidentifiedandcharacterizedthelongchainbranchingcomponentoflowdensityPE.36Thisiscriticaltoimprovedunderstandingofeffectsoflongchainbranchingontherheologicalandprocessingpropertiesofpolyolefins.35,36TheporeAnalyticalChemistry,Vol.xxx,No.xx,MonthXX,XXXX

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sizeoftheceramicmembranedeterminedthelowerMWlimitof50kDa.ContinuedmembraneadvancementssuchassmallerporediametersandexpandedsolventcompatibilitywillopenmoredoorsforcharacterizingorganosolublemacromoleculesbyFlFFF.LOOKINGTOWARDSTHEFUTURE

FFFhascomeofageasamainstreamanalyticaltechniqueforseparatingandcharacterizinganalytespeciesthatrangeinsizefromthelownanometerstotensofmicrometers.TheNationalInstituteofStandardsandTechnologyhasusedFFFtosupportthedevelopmentofnewcertifiedreferencematerials.TheFoodandDrugAdministrationlistsFFFamongthecommontechniquesusedtocharacterizenanomaterials(ManualofPoliciesandProcedures5015.9).Asthenumberofapplicationsandpublicationsrise,methodsdevelopmentisgivingwaytomethodsrefinement,therebymakingtheimplementationofFFFalesstimeconsumingtaskforusersnewtothefield.ThenumberofpotentialFFFapplicationsseemsvastasnewnanomaterialsaredesignedandsynthesizedandnewmixturesandhybridsareintroduced.NewchallengeswillariseastheapplicationsofFFFinstrumentsmovebeyondtheinstru-ments’originaldesignpurpose.AnexampleistheFlFFFsepara-tionof<10nmquantumdotssuspendedinorganicsolvents,forwhichsuitablemembranesarenotcurrentlycommerciallyavail-able.Movingfromanalyticalscaletopreparativescaleapplicationsisalsoapracticalchallenge.Forexample,typicalFFFexperimentsampleinjectionvolumesaretensofmicroliterscontaining∼106-108nanoparticles.Thoughthisprovidesgoodmeasure-mentstatistics(relativetomicroscopy),thisremainsintherealmofanalyticalscale.Preparativescaleseparationsthatcouldproducesuitablequantitiesofuniformsamplesfortestingwouldgreatlyextendunderstandingofnano-sizedmaterialpropertiesandtheirrelationtoproductperformance.ThetimeisalsoripeforminiaturizationbecausecombinedmicrofluidicsandFFFknowledgecouldproducedeviceswithimproveddiagnosticoronlineprocessingcapabilities.GrowinganalyticalneedsthatcanbeaddressedbynewapplicationsofFFF-basedinstrumentationwithdifferentfieldsandcapabilitieswillcontinuetodrivedevelopmentinthisfield.

ACKNOWLEDGMENT

TheauthorsgratefullyacknowledgesupportfromtheNationalScienceFoundation(CHE-0515521,CHE-1013029,CBET-0968042,andDMR-0820518).A.AshamesissupportedbytheLibyanMinistryofEducationandScientificResearch.S.KimR.WilliamsisanAssociateProfessorofChemistryandtheDirectoroftheLaboratoryforAdvancedSeparationsTechnologiesatColoradoSchoolofMines.ResearchintheWilliamslabfocusesonthedevelopmentandapplicationofseparationandcharacterizationtechnologiesforanalytesthatspannanometertomicrometersizes.Dr.J.RayRunyonisapostdoctoralresearcherattheColoradoSchoolofMines.HisresearchfocusesonthedevelopmentofnovelFFFandlightscatteringapproachestocharacterizecomplexpolymersandnanoparticlessystemsandascertainstructure-property-performancerelationships.HereceivedhisPh.D.in2009undertheguidanceofProf.Williams.AkramAshamesisaPh.D.graduatestudentintheWilliamsresearchgroup.WithaB.Sc.inpharmacyfromAlfatehUniversity,Libya,hisresearchfocusesondevelop-ingandimplementingFFFwithdifferentdetectionsystemsforthepreparationandstudyofnanocarriersfordrugdelivery.Addresscor-respondencetoWilliamsatLaboratoryforAdvancedSeparationsTech-nologies,DepartmentofChemistryandGeochemistry,ColoradoSchoolofMines,Golden,CO80401;krwillia@mines.edu;Fax:1-303-273-3629.H

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