In the present proof-of-concept study, we demonstrate that retention time, selectivity and resolution can be increased in asymmetrical flow field-flow fractionation (AF4) by introducing microstructured ultrafiltration membranes.
Trang 1jou rn al h om ep a g e : w w w e l s e v i e r c o m / l o c a t e / c h r o m a
fractionation
Maria Mariolia,∗, Ü Bade Kavurtb, Dimitrios Stamatialisb, Wim Th Koka
a Analytical Chemistry Group, van’t Hoff Institute for Molecular Sciences, University of Amsterdam, P.O Box 94157, 1090 GD Amsterdam, the Netherlands
b (Bio)artificial Organs, Department of Biomaterials Science and Technology, TechMed Institute, University of Twente, P.O Box 217, 7500 AE Enschede, the
Netherlands
a r t i c l e i n f o
Article history:
Received 10 April 2019
Received in revised form 25 June 2019
Accepted 2 July 2019
Available online 3 July 2019
Keywords:
Field-flow fractionation
Flow over grooves
AF4
Computational fluid dynamics
Microstructured membranes
Protein separation
a b s t r a c t
Inthepresentproof-of-conceptstudy,wedemonstratethatretentiontime,selectivityandresolution canbeincreasedinasymmetricalflowfield-flowfractionation(AF4)byintroducingmicrostructured ultrafiltrationmembranes.Evenlyspacedmicron-sizedgrooves,thatareplacedperpendiculartothe channelflowontheaccumulationwallofafield-flowfractionationsystem,causeadecreaseinthe zonevelocitywhichisstrongerforlargersolutes.Thishasbeendemonstratedinthermalfield-flow fractionation,andweprovethatthisisalsothecaseinAF4.Weexaminethehypothesistheoretically andexperimentally,bybothcomputationalandphysicalexperiments.Bymeansofmomentanalysis,we derivetheoreticallyasetofequationswhich,undercertainconditions,describethemasstransportand relateretentiontime,selectivityandplateheighttothedimensionsofthegrooves.Physicalexperiments arecarriedoutusingmicrostructuredpolyethersulfonemembranesfabricatedbyhotembossing,andthe experimentalresultsarecomparedwithcomputationalfluiddynamicsexperiments
©2019TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
1 Introduction
Asymmetrical flow field-flow fractionation (AF4), the most
appliedsubtechniqueofthefield-flowfractionation(FFF)family,
isanestablishedanalyticalmethodtoseparatemacromolecules
andnanoparticlesaccordingtotheirhydrodynamicsizeundermild
conditions[1–3].Thecouplingwithvariousphysicalandchemical
detectorshascontributedsignificantlytoitspopularityasitcan
providevaluableinformationsuchasmolecularweight
distribu-tion,sizedistribution,conformationandchemicalcompositionin
asinglerun[4 Considering therapidgrowthinbiotechnology,
nanotechnologyandpolymerengineering,itisevidentthatAF4is
goingtowitnessafurthergrowthinapplicationsinthecoming
years.Inthisregard,itisworthwhiletoproposeandinvestigate
possiblenewtechnicaldevelopmentsthat mayimprove
perfor-mance
Inthisstudyweinvestigatethepossibilityofincreasing
reten-tion time, selectivity and resolution by using microstructured
ultrafiltration(UF)membraneswithparallelgroovesontheir
sur-∗ Corresponding author.
E-mail address: M.Marioli@uva.nl (M Marioli).
face (Fig 1) However, considering that AF4 is a very flexible techniquewhere severalparameterscanbealtered tooptimize separation,firstajustificationshouldbegivenfortheusefulness
ofsuchadevelopment
AccordingtotherigorousFFFtheory,theretentiontimeof well-retained(withretentionratio<0.1)componentsinAF4isequal
to[5
tR= w2 6Dln
1+ V˙c
˙Vout
B
(1)
where wisthechannelthickness, ˙Vcthecross-flowrate, ˙Voutthe channeloutletflow rateandBthefractionoftheaccumulation areaafterthefocusingpoint.Therefore,theselectivityofapairof well-retainedsolutesequalstheratiooftheirdiffusioncoefficients,
˛=tR,2
tR,1 =D1
D2
(2)
andconsequently,itcannotbealteredbychangingthe experimen-talparameters.Resolutioncanbeimprovedbyreducingtheplate https://doi.org/10.1016/j.chroma.2019.07.001
0021-9673/© 2019 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).
Trang 2Fig 1.AF4 with microstructured membranes.
heightwhich,basedonthenonequilibriumtheory(for<0.1),is
equalto[6
H= 24D2v0
whereucr isthecross-flowvelocitythoughtthemembrane and
v0 is thecross-sectional mean carrier velocity Hence, a high
cross-flowvelocitydecreasesplateheight.However,itmaylead
toadsorptiononthemembrane and massoverloading for
sen-sitivemacromolecules.Inaddition,highflowratesarehindered
bythetransmembranepressurewhenultrafiltration(UF)
mem-braneswithverylowmolecularweightcut-off(MWCO)areused
toseparatesmallmacromolecules
Thesolutescanberesolvedatlowercross-flowratesby
increas-ingtheretentiontime,sinceaminimumtimeisrequiredtoachieve
separation [7 which couldbe accomplished by increasingthe
cross-flowtooutlet flowratioor thespacerthickness[8 Very
highcross-flowtooutletflowratiosareimpractical,particularly
forUFmembraneswithlowMWCO,andmaydistorttheparabolic
flowprofile[9 Inaddition,theuseofathickerspacerresultsin
higherrequiredfocusingtimesand moredilutionwitha
subse-quentdecreaseinsensitivity[7 Moreover,alowaspectratiob/w
(<30),wherebis thechannel breadth,mayaggravateedge and
endeffectsincreasingplateheightandreducingrecovery[10,11]
Therefore,itcouldbebeneficialtoinvestigateamethodthatcould
increase retention and resolution without altering the optimal
cross-flow,spacerthicknessandcross-flowtooutletflowratio
Theconceptofanaccumulationwallwithmicron-sizedgrooves
inFFFhasbeenintroducedin1978byGiddingsetal.[12]asan
attempttoincreaseretentionforsmallanalytesinthermal
field-flowfractionation(ThFFF).Inaddition,groovedsurfaceshavebeen
incorporatedinmicrofluidicchannelsforvariousotherapplications
suchastoenablemixing[13]andtoseparatecellsand
micropar-ticles[14].Navierhasdescribed thatmacroscopicallytherough
surfaceisequivalenttoasmoothsurfacewithpartialslip[15–17]
Infact,forthisreason,asmallslipmightexistontheflatmembrane
oftheAF4channel,asa resulttotheporosity,but itis
negligi-bleforUFmembranes[18].NanostructuredUFmembraneshave
beenfabricatedbynano-imprintinglithography[19–21], where
membraneswerehot-embossed,andmicrostructuredpolymeric
materialshave been developed withphase separation [22–24],
whereapolymersolutioniscastoverapatternedmold
Thescopeofthisstudyistoconductaproof-ofconcept
inves-tigationtoassesstheeffectofmicrostructuredmembranesonthe
retentiontime,selectivityandresolutioninAF4.Ahotembossing
methodwaschosenforthefabricationofthesemembranes.We
sharefundamentaltheoryandexperimentalfindingsthat
comple-mentandexpandthepreviousstudywithperpendiculargrooves
inThFFF[12]
Fig 2. Left-hand figure: display of the theoretical model Right-hand figure: velocity profile (a) over a flat membrane and (b) over a grooved membrane where the velocity zero-plane is taken on the edge of the ridge (x = h).
2 Theory
2.1 Transportequationsandmomentanalysis Here,wedescribeasimplifiedmodelthatenablesustoderivean analyticalsolutiontotheproblemofmassmigrationovergrooves
inanAF4channel.Inthismodelthegroovesareformedby zero-widthridgeswithauniformheighthonthemembranesurface, perpendiculartotheflowdirection.Slipflowthroughthegrooves
isneglected;thezero-velocityplanefortheaxialflow(v)istaken
atthetopoftheridges(Fig.2)
Thefollowingsimplificationshavebeenmade:
(1)Moleculardiffusionintheaxial(z-)directionisneglected (2)Thedevelopmentoftheconcentrationprofileinthe perpen-dicular(x-)directioniscompletebeforeelutionisstarted,bya precedingfocusingstepintheprocedure
(3)Onlywell-retainedcompoundsareconsidered(withretention ratio <0.1) Such compounds are present predominantly closetotheaccumulationwall,wherethelinearpartoftheflow profileprevailsandthecross-flowvelocityucrmaybe consid-eredasbeingequaltothefluidvelocitythroughthemembrane For well-retainedcompounds,themathematicscanbe sim-plifiedsinceintegralsover theheightofthechannelcanbe takenfromx=0toinfinityinsteadoftotheupperwallposition (x=w),withgoodaccuracy
(4)Therearenointeractionsbetweentheproteinandthe mem-brane
(5)Flowconditionsarelaminar.Thisassumptionshouldholdtrue sincethepresenceofperpendiculargrooves,whicharesmall compared tothechannelthickness,reduceslocallytheflow velocity and decreasestheReynolds number [16] Although eddiesmayexistinthecornersofthegrooves,theflowvelocity
isverylowthereandthefluidisalmoststagnant
Thetransportofacompoundi,withalocalconcentrationci=
ci(x,z,t),isgivenbythesimplifiedgeneraltransportequation
∂ci
∂t =Di∂2ci
∂x2 +ucr∂ci
∂x −v(x)∂ci
whereDiisthediffusioncoefficientofthecompoundofinterest, andv(x)thelocalaxialflowvelocity.Themoleculardiffusionterm alongthez-directionisneglectedintheRHSofEq.(4).Theplus signfor thesecondtermoftheRHSappearsbecausea positive valueistakenforucr,evenwhenthecrossflowisinthenegative
x-direction.Theassumptionthattheanalytehasbeenintroduced
inthechannelasafiniteplugleadstotheboundaryconditions
Trang 3forthecompoundto
Di∂ci
∂x +ucrci=0 for x=0,w (4b)
Twosetsofmomentsaredefined.Localmoments,thatdescribe
themassdistributionofacompoundiinafluidlayeratacertain
distancexfromthemembrane,aredefinedas
mn,i(x,t)=
+∞
−∞
andoverallmoments,thatdescribethemassdistributioninthe
axialdirectionintegratedovertheheightofthechannel,as
Mn,i(t) =
w
0
mn,i(x,t) dx≈
0
mn,i(x,t) dx (6) MomentsexistwhentheintegralsconvergeinEq.(6),i.e.,whenit
canbeassumedthattheconcentrationofacompoundiapproaches
zerofastenoughwhenzgoestoplusorminusinfinity.Thiswillbe
thecasewhenthecompoundwasintroducedinthechannelasa
plugorpeakoffinitewidth
WhenbothsidesofthegeneraltransportEq.(4)aremultiplied
withznandintegratedoverzfromminustoplusinfinity,
expres-sionsareobtainedforthelocalmomentsofi
∂mn,i
∂t =Di∂2mn,i
∂x2 +ucr∂mn,i
∂x +nv(x)mn−1,i (7) ThethirdtermoftheRHSinthisequationisobtainedbypartial
inte-grationwiththeassumptionthatznci(x,z,t)vanishesforz→±∞
Whenalocalmoment(n−1)isknown,thisequationcanbeusedto
evaluatethenextlocalmoment(n).IntegrationofEq.(7)overthe
heightofthechannel,consideringboundarycondition(4b),gives
anexpressionfortheoverallmomentMn,i
∂Mn,i
∂t =n
0
2.2 Thezerothmoment(massdistribution)
IntegratingbothsidesofEq.(4)overzfromminustoplusinfinity
givesanexpressionforthelocalzerothmomentofi,
∂m0,i
∂t =Di
∂2m0,i
∂x2 +ucr∂m0,i
Undertheassumptionthatfocusingwascomplete,andasteady
statewasreachedbeforetheexperimentwasstarted,bothsidesof
Eq.(9)mustbezero,andthemassdistributionovertheheightof
thechannelcanbefoundas
m0,i=m∗0,i·exp
−ucr
Dix
(10)
wherem∗0,iisthezerothmomentonthemembranesurface(with
x=0).Theexp.concentrationprofileextendsoutfromtheupper
wall(x=w)here Eq (10)describesthewell-known
exponen-tialconcentrationprofileontheaccumulationwallinFFF,witha
characteristiclayerthickness
equaltoDi/ucr.Whenthe con-centrationoftheanalyteisscaledsoas
m*
0,i= ucr
Di
(11)
theoverallzerothmomentM0,ibecomes1,andthehigheroverall
momentsareautomaticallynormalized
2.3 Thefirstmoment(meanretentiontime) Themodelforthegroovedsurfaceusedhere(Fig.2 witha stagnantlayerof fluid determinedby thegrooveheight h,and approximatelylinearlyincreasingchannelflowratefromtheslip planeatthetopoftheridges,givesforthelocalaxialflowvelocity
v(x)=6(x−h)
WhenEqs.(10)–(12)aresubstitutedintoEq.(8),
∂M1,i
∂t =
h
6(x−h)
w vucr
Di
exp
−ucr
Dix
theaxialvelocityviofthecompoundisobtained(withforsimplicity integrationtoinfinityinsteadoftox=w),
vi=∂M1,i
∂t = 6Di
ucrwvexp
−ucrh
Di
(14)
Eq.(14)withh=0givesthewell-knownexpressionforthezone velocity over a flatmembrane With a groovedmembrane, the velocitydecreasesexponentiallywiththeratiooftheridgeheight overthecharacteristiclayerthickness.Fortheretentiontimethe oppositecanbewritten
tR,i=tR,iFLexp
+ucrh
Di
(15) wheretFL
R,iistheretentiontimewithaflatmembrane,under oth-erwise thesameconditions Theretention time increasesmore stronglybythepresenceofthegroovesforcompoundswithasmall layerthickness,i.e.,formorestronglyretainedcompounds
In the separation of two components, the selectivity ˛ is increasedwithincreasingridgeheightanditcanbewrittenas
˛=tR,2
tR,1 =D1
D2
exp
ucrh
1
D2 − 1
D1
=˛FLexp
h
1
˛FL−1 (16) where˛FListheselectivitywithaflatmembrane,and1the char-acteristiclayerthicknessofthefirst,leastretainedcompound.In Fig.3a,thecalculatedeffectofthe(relative)heightofthegrooveson theretentiontimesandtheselectivityisshownfortwocompounds withdiffusioncoefficientsthatdifferbyafactorof√2.
2.4 Thesecondmoment(peakvariance)
Toevaluatetheinfluenceofthegroovedsurfaceonpeak broad-ening,first anexpressionfor thedevelopmentofthelocalfirst momentshastobederived.Inthis,wefollowtheapproachtaken
byTaylorandArisintheirtreatmentofpeakbroadeningin cylin-dricalchannels,andinearlyworkofGiddingsondispersioninFFF [25].TheyfoundsolutionsforthegeneraltransportEq.(4)inthe formofasumoftransientfunctionsandastationaryfunction.The transientfunctionsdescribetheconcentrationchangesintimeand spacedirectlyafterthestartofthe’elution’andtheydependonthe initialconditions.Itwasshownthatthesetransientfunctionsdie outrapidly,andthatastationarysituationdevelopsinwhichthe localcentersofgravityatdifferentdistancesfromthewallare situ-atedinasteadyprofilearoundtheoverall(mean)centerofgravity
ofthetransportedplugofthecompoundofinterest.Here,a solu-tionissoughtforEq.(4)describingonlythestationarysituation, i.e.,asolutionthatobliges
1
m (x)
∂m1,i(x)
∂t = ∂M1,i
∂t =vi for all x (17)
Trang 4Fig 3.Theoretical estimation of variables as a function of the relative ridge height: a)
increase in selectivity and retention time for two solutes with diffusion coefficients
that differ by a factor of √
2 (e.g., monomer and dimer); retention times here are normalized with the retention time of the smaller solute for a flat membrane t FL
R,1 b) increase in plate height c) increase in resolution.
Asetofparticulatesolutionsform1,i(x)canbefoundthatsatisfyall
boundaryconditions
For0≤x<h,
mA1,i(x,t) = 6v
w
t− Di
u2 cr
− x
ucr
exp
−ucrh
Di
exp
−ucrx
Di
(18a) andforx≥h,
mB1,i(x,t) = 6v
w {(x−h)
2
2Di +x−h
u +
t− Di
u2 cr
− x
ucr
exp
−ucrh
Di
}exp
−ucrx
Di
(18b)
Theincreaseintimeoftheoverallsecondmomentcannowbe
foundbysubstitutingEqs.(12b)and(18b)intoEq.(8)
∂M2,i
∂t =2
6v0
Sincethefluidvelocityiszerofor0<x<h,Eq.(18a)doesnothave
tobeincludedintheintegration.Centralizingoftheoverallsecond momentgivestheincreaseofthespatialvarianceintime
∂2 z
∂t =∂M2,i
∂t −2M1,i(t)∂M1,i
andfinally,theplateheightHcanbeobtainedas
H= ∂z2/∂t
ThefinalresultforHis
H=24D
2
iv0
u3
crw
5
2exp
+ucr
Dih
−32− ucr 2Dih exp
−ucr
Dih
(22)
Foraflatmembrane,withh=0,thesecondandthirdfactorsinthe RHSofEq.(22)areequalto1,andthewell-knownexpressionfor
H(HFL)isobtained(Eq.(3))
InFig.3 theincreaseoftheplateheightwiththerelativeridge heightisshown,andinFig.3ctheincreaseinresolutionoftwo soluteswithratioofdiffusioncoefficients√2isshown.Weobserve thatforgrooveheighth=1.51,thereisatwo-foldincreasein res-olutionandafour-foldincreaseintheretentiontimeoftheless retainedcomponent.Forcomparison,thesameincreasein reso-lutioncouldbeachieved (withoutaltering thecross flow)by a two-foldincreaseofthespacerthicknessorapproximatelyten-fold increaseofthecross-flowtooutletflowratio
3 Materials and methods
3.1 Samplesandcarriereluent Bovineserumalbumin(BSA),␥-globulin,apoferritin, thyroglob-ulinandhemoglobinwerepurchasedbySigma–Aldrich(MO,USA) PBS0.15M(20mMduetosodiumphosphatesalts)withapHof 7.2wasusedasacarriereluentfortheAF4experimentsandas
adiluentfortheproteins.Allproteinsampleswerepreparedata concentrationof1mg/mL
3.2 Fabricationandcharacterizationofthemicrostructured(MS) membranes
Twosiliconmolddesignswithparallelgrooveswereusedfor preparationofthemicrostructuredmembranes.MoldI(LioniXBV, TheNetherlands)hadapatternedareaofdiameter15.1cmwith groovesof cavity widthc=50m,ridge width r=50m, ridge heighth=12mwhereasMoldII(MESA+cleanroom,Universityof Twente,TheNetherlands)hadapatternedareaofdiameter6.8cm withgroovesofc=30m,r=20mandh=25m Polyethersul-fone(PES)membraneswith10kDaand30kDamolecularweight cut-off(MWCO)(Sartorius,Germany)wereusedforthemembrane patterningwithoutanypretreatment
Microstructured (MS) membranes were prepared via hot embossing which was performed with an imprinter (Obducat, Sweden)inMESA+cleanroom(UniversityofTwente).The emboss-ingtemperature,pressureandtimewere120◦C,40barand180s, respectively and demoldingoccurredat 40◦C [20] Surface and cross-section images of the microstructured membranes were takenby scanningelectron microscopy(SEM) equipment,XL30 ESEM-FEG(Philips,TheNetherlands)orJEOLJSM-6010LA(JEOL, Japan).MSmembraneI(Fig.4a)wasfabricatedbyhot-embossing
a PES 10kDa membrane with the Mold I, and MS membrane
II(Fig.4b)byhot embossinga PES30kDa membrane withthe MoldII.Membrane sampleswerewashed,dried,broken in liq-uidnitrogenforcrosssectionimagesandgold-sputteredforSEM imaging
Trang 5Fig 4.Microstructured membranes and AF4 channels: a) MS membrane I (hot embossed with Mold I) and Channel I b) MS membrane II (hot embossed with Mold II) and Channel II.
Cleanwaterflux(Jw)valuesofthemembranesweremeasured
withdead-endAmiconStirredCell(Model8050,MerckMillipore,
MA,USA)andultrapurewater(MilliQsystem,MerckMillipore)
Measurementswereperformedatfourdifferenttransmembrane
pressures(P)in therange of0.5–2bar,afterremoving ofthe
membrane preservativesby immersingin water and after
pre-compactionat2bar.Theweightofpermeatedwaterversustime
wasmeasuredandthecleanwaterflux(JwinL/m2/h)was
calcu-latedforeachpressureconsideringtheeffectivemembranesurface
area,whichwas13.4cm2 (Theareaisassumedasconstantafter
preparationofamicrostructuredsurface).Thecleanwater
perme-ance(CWP,inL/m2/h/bar)ofthemembranewasdeterminedfrom
theslopeofJwversusPrelationship
3.3 AF4experiments
TheAF4systemwasanEclipseDualTecsystem(Wyatt
Technol-ogyEurope,Germany)connectedtoanAgilentHPLC1200system
(AgilentTechnologies,Germany)thatconsistedofadegasser,an
isocraticpump,aUVdetectorandanautosamplerequippedwith
athermostat.Thetemperatureoftheautosamplerwassetat5◦C
TwoAF4trapezoidalchannelswereused,designatedasChannel
IandII,oneforeachmembrane/moldsize(Fig.4).TheMS
mem-braneswerecutwiththegroovesperpendicularandintheshape
oftheporousfritwithsurgicalscissors
ChannelIwasacommercialAF4channel(WyattTechnology
Europe) which was used with the largerpatterned membrane
(d=15.1cm).Ithadtip-to-tiplength13.3cmandaccumulationarea
15.6cm2(Fig.4a).Thenominalspacerthicknesswas250or350m
Thefocus-flowwas1.5mL/minfor3minandthefocusingpointwas
setat18%ofthechannellength.Theinjectedvolumewas10L
(10ginjectedmass)andtheUVdetectionwasat280nm
ChannelIIwasaminiaturizedchannelcreatedtotestthesmaller
patternedmembrane(d=6.8cm).It hadtip-to-tiplength6.3cm
andaccumulationarea7.24cm2 (Fig.4b).Itwascreatedusinga
commercialchannelmodifyingitsupperinlayandspacer.Inthe
upperinlaytwointernalthreadsweremilledtoconnectthetubing
fittingsfortheinletandoutlet.Thespacerwasfabricatedcutting
MylarA4sheetsofnominalthickness250and350m.The focus-flowwas0.8mL/minappliedfor3minandthefocusingpointwas setat18%.Theinjectedvolumewas5L(5ginjectedmass)and
UVdetectionwasat220nm
3.4 Computationalfluiddynamics(CFD)
Afiniteelementsolver,COMSOLMultiphysics5.2(COMSOLInc.,
MA,USA),wasusedtomodeltheAF4channelandsimulatethe proteinmigrationovertheflatandthepatternedmembrane.To reducethemodelintotwo dimensionsforlowercomputational cost,asymmetricalchannelwasmodelledinsteadofan asymmet-rical.Forthispurpose,asimplerectangulardomainwascreated, withaflatorgroovedbottomboundary.Ameshoffreetriangular elementswascreatedwithveryfineelements(<1m)inthe prox-imitytothebottomboundarytosimulateproteinmigrationwith highaccuracy
Todescribetheflow,laminarflowofanincompressiblefluid wasusedand theboundaryconditions(inlets,outlets)wereset
todefinechannel flowandcross-flowvelocities(itwasverified laterfromtheresultsthattheassumptionofthelaminarflowwas validbythecellReynoldsnumber).Thecross-flowvelocitywas dis-tributedhomogeneouslyalongthebottomboundary(membrane) Theoption“transportofdilutedspecies”(includingconvectionand diffusion)wasusedtosimulateproteinmonomeranddimer.The studyoftheflowprofilewassolvedasasteadystateproblemand theoutput(velocityfield)wasusedtosolvethetimedependent problemoftheproteinmigrationwithaBDF(Backwards Differen-tialFormula)solver.Therelativeandtheabsolutetoleranceswere setat10−4.Theinitialandthemaximumtimestepswereset0.001s and0.5s,respectively
4 Results and discussion
4.1 Characterizationofthemicrostructuredmembranes Themicrostructuredmembranes,designatedasMSmembrane
Iand IIhadsimilarridgeheight,h∼12m,anddifferent
Trang 6peri-Table 1
Protein recovery in AF4 before and after hot embossing of the UF membranes AF4 conditions: ˙V c = ˙V out = 1 mL/min.
Recovery (%) ± s.d.
odicity(i.e.,thesumofcavityandridgewidth),100and 50m
respectively (Fig 4) The shape of the patterns was
rectangu-lar with round corners (MS membrane I) or ellipsoidal (MS
membrane II)as therectangularcavities of themoldwere not
completely filled during embossing Although the polymer is
heatedaboveits glasstransitiontemperaturein hot embossing
processes,embossing wasperformed belowtheglasstransition
temperature, since collapse of the pores and loss of
perme-ance are reported in the literature for a PES membrane [20]
TheCWPs ofthe non-patterned membraneswereestimated as
150±20L/m2/h/bar (for membranes with 10kDa MWCO) and
271±113L/m2/h/bar (formembraneswith30kDaMWCO) The
CWPsofbothmembranesdecreasedafterhotembossing;MS
mem-braneIhadCWPof74±1L/m2/h/barandMSmembraneIIhadCWP
of130±18L/m2/h/bar
ProteinrejectionoftheMSmembraneswasevaluatedwiththe
AF4system;therecoveryoffourproteinsofdifferentmolecular
weight(66.5–669kDa)wasestimatedfromtheratioofthepeak
areaof thefractionatedsampletothepeak areaofthe
unfrac-tionatedsample.Thepeakareaoftheunfractionatedsamplewas
estimatedfromthefractogramobtainedbyinjectingandeluting
thesameamountoftheproteinwiththesamechanneloutletflow
rate,withouttheapplicationof focusflow orcross-flowexcept
forapoferritin.Thesolutionofapoferritincontainedlow
molec-ularweightcomponentswhichwereUV-activeatthedetection
wavelength,andthereforefocusflowwasappliedfortheirremoval
Forthisreason,therecoveryvaluesofapoferritinmaybeslightly
overestimatedforallmeasurements(bothwithflatandwithMS
membranes).TheexperimentalresultsaregiveninTable1;the
recoveryofthesmallerproteins(BSAand␥-globulin)was
signifi-cantlylowerfortheMSmembranes
Thedecreaseinrecoveryafterhotembossingshouldindicate
anincreaseintheactualMWCOratherthanproteinadsorption
sincethePESmembranesusedinthisstudyarehydrophilicwith
lowfoulingpropertiesforproteinsolutions.Thiswasconfirmedby
injectingandfocusingforseveralminutesahighvolume(100L)
ofaconcentratedsolution(30mg/mL)ofhemoglobin(∼65kDa)
whichhasaredcolor.Itwasobservedthatthesamplewasfocused
asanarrowbandwithaflatmembranewhileitwaspassingthrough
thecross-flowwithanMSmembrane.Whenthemembranewas
removedandvisuallyinspected,itwasnotstainedwhichwould
indicateadsorption
Theaforementionedresults(increaseinMWCOanddecrease
inCWP)seemcontradictingsincelowerCWPisoftencorrelated
withadecreaseinthesizeornumberoftheporesoftheselective
(patterned)side.ApossibleexplanationisthattheCWPdecreases
becauseof the membrane compaction (particularly in thearea
ofthegrooves’valleyswhichexperiencethehigheststress
dur-inghotembossing).Inaddition,theincreaseintheactualMWCO
mightberelatedtoanincreaseoftheporesizeof thegrooves’
ridgesbecause of themembrane deformation or toother local
defectsthatoccurduringimprinting/demoldingwhichare,
how-ever, small enough to affect only the recovery of the smaller
proteins
Incontrastwithourobservations,Marufetal.[20]showedthat
hotembossingcouldleadtosimilarCWPand lowerMWCOfor
anotherPESmembraneandamoldpatternedwithsmallergrooves (inthesub-micronrange).Perhapstheporedeformationtherewas minimalbecauseofthesmallersizeofthegrooves.However,the effectofthemembranecompactionontheCWPandthedifference
inthestressdistributiononthevalleysandontheridgesduring hotembossinghavebeendiscussedinthesestudies[21].Overall ourresultsindicatethathot-embossingneedstobeoptimizedto avoidchangesoftheMWCOsincetheconceptwouldbe benefi-cialparticularlyforlowmolecularweightanalytes,andingeneral
UF membraneswithhighsolventpermeability are preferredin AF4
Using BSA as thecalibrant withknown diffusioncoefficient (6.21·10−11m2/s[26]),theactualchannelthicknessforthe Chan-nel I and the Channel II witha flat membrane was estimated
305±6mand294±8mrespectively,andthediffusion coef-ficientofapoferritinwasestimated3.38·10−11m2/sfromEq.(1) Thesevalueswereusedinthesimulations.However,MS mem-branesarealreadycompressedduetohotembossing,andhence anyadditionalcompressioncausedbythespacerisexpectedto
besmall.Thiswouldresultinlargeractualchannelthicknessand consequentlyin longerretention times Thedifference in com-pressionbetweentheflatandtheMSmembraneswasevidentby visualinspectionwhenthemembraneswereremovedfromthe channelandinspected.Unfortunately,themethodwithaprotein
ofknowndiffusivitycannotbeappliedfortheMSmembranesas theretentiontime increasesbythepresenceofthegroovesfor well-retainedcompounds.However,inorder toassesscorrectly theeffectofgrooves,theactualchannelthicknessoftheMS mem-branes needs tobe measuredand we attempted this by other means
First,themembranecompressibilitywasestimatedfromthe dif-ferenceinthethicknessofthecompressedandnon-compressed partofthemembranes,measuredbySEMandamicrometerscrew gaugewhentheywereremovedfromthechannel.The compres-sionthatoccurredwithaflatandaMSmembranewas∼50m and∼20mrespectively.Thiscorrespondsto11%largerchannel thicknesswiththeMSmembranes.However,thesemethodshave lowprecisionsincetheymeasureonlyaverysmallpartofthetotal membraneareawhenthemembranesaredry.Second,weapplied therapidbreakthroughmethod[27]inthefractogramsobtained fortherecoveryexperimentswithinjectionand elutionof thy-roglobulin(withouttheapplicationoffocusorcross-flow).Thevoid volumewasmeasured13%largerwiththeMSmembranewhich correspondstoa13%thickerchannel.Thisresultisinclose agree-mentwiththefirstmethod.However,bothmethodsdonotuse cross-flowwhichmightslightlyaffectthemembranecompression and/orswelling
4.2 AF4experiments Apoferritinandthyroglobulinwerechosenasthemodel pro-teinstoassesstheeffectofthegroovesonretentiontime,selectivity andplateheight,sincetheyexhibitedhighrecoverieswiththe pat-ternedmembranes(Table1).InFig.5thefractogramsofapoferritin, obtainedusingflatandMSmembranes,areoverlaidafter subtrac-tionofthetimethatwasrequiredforthefocusingstep.Forboth
Trang 7Fig 5. Comparison of flat and MS membranes analyzed with flow rates ˙V c = ˙V out = 1.0 mL/min for a) Channel I and MS membrane I and b) Channel II and MS membrane II.
Fig 6.CFD model for the Channel II/MS membrane II system: a) Mesh of the model in the beginning of the channel, b) velocity profile over the grooves, c) concentration profile of apoferritin over the grooves and d) derived concentration at the outlet (right boundary) for every time point for the monomer and dimer.
Trang 8Table 2
Comparison of flat and MS membranes for both channels with respect to the plate height of the monomer H, the retention time of the monomer t R,1 and the selectivity a between the monomer and the dimer The error bars are given at 1 level and reflect the membrane-to-membrane reproducibility.
Channel I
Flat membrane,
w = 350 m
MS membrane I,
w = 350 m
MS membrane I,
w = 250 m
Channel II
Flat membrane,
w = 350 m
MS membrane II,
w = 350 m
MS membrane II,
w = 250 m
channels/MSmembranesystemsandthesamespacerthicknessof
350m(Fig.5left-handfigures),thereisaconsiderableincrease
inretentiontime,selectivityandresolutionbetweenmonomerand
dimer.Althoughthereismorepeakbroadeningwiththepresence
ofthegrooves,resolutionishigherbecauseofthehigherselectivity
(asexpectedbythetheory,Fig.3).Consequently,thesame
resolu-tioncouldbeachievedwiththeMSmembranesand applyinga
lowercross-flowrate,oralternativelyusingathinnerspacer(Fig.5
right-handfigures)
Therefore,theMSmembranescouldbebeneficialasthesame
retentionandresolutioncouldbeachievedwithlowercross-flow
rates without the need to increase spacer thickness or to use
impracticalcross-flowtooutletflowratios.Inpracticethatwould
beparticularlyusefulforrelativelysmallsolutes(suchasBSAor
evensmaller)sincelargersolutescanbeanalyzedwithoptimal
spacerthicknessandflowrates,andthereforethereisaneedto
fabricateMSmembraneswithlowerMWCO.Thechallengesand
theprocedurestooptimizeAF4methodsforsmallsoluteswitha
300DaMWCOmembranehavebeenreported[28].Smaller
macro-moleculesaretypicallyanalyzedbysizeexclusionchromatography
(SEC)wheretheyexhibitverygoodresolution,butinsomecases,
SECisnotsuitable,forinstance,whenthereisstrongnon-specific
adsorptioninthechromatographicsupportandwhenlarge
macro-moleculesco-existinthesamplethatneedtobeanalyzed.Inthe
lastcase,across-flowprogramwithexponentialdecayshouldbe
usedasthegrooveswouldcauseverystrongretentionforthelarge
components
Aseriesofexperimentswerecarriedoutusingdifferent
cross-flowrateswhileretainingtheratio( ˙Vc/ ˙Vout=1);theresultsare
displayedinTable2.Theplateheightofthemonomerwas
esti-matedfromthewidthathalfpeakheight.Anumberofconclusions
maybedrawnfromtheseexperimentalresults.Althoughthereis
alargedepartureoftheengineeredgroovesfromthetheoretical
model(i.e.,noslip,infinitesimalridgeandrectangularshape),the
underlyingconclusionswerefoundsimilar
First,fromTable2,itcanbeseenthatforhigher ˙Vcandsame
˙Vc/ ˙Vout the retention time of the monomer and theselectivity
betweenmonomeranddimerweresimilarfortheflatmembranes
asexpectedbythetheory(Eqs.(1)and(2))butincreasedfortheMS membranes.Thisisinlinewiththetheoreticalequationsderived
bymomentanalysis(Eqs.(15)and(16)).Secondly,forthesame experimentalconditions,theincreaseinselectivitywashigherfor thyroglobulin(lower)anditwasindependentofthespacer thick-ness,aspredictedbythetheory.Lastly,theincreaseinretention timeandselectivityforthesamecross-flowvelocitywashigherfor theMSmembraneII,probablyduetothesmallerslipbecauseof thesmallerperiodicityofthegrooves
It is howeverimportant tonotethat partof theincrease in retentiontime isa resultofthelargeractualchannelthickness withtheMSmembranes.Asitwasmentionedabove,thechannel thicknesswasestimated∼12%largerwithMSmembranes,which correspondsto∼25%longerretentiontimescausedbytheeffectof themembranecompression,astheretentiontimeisproportional
tow2(Eq.(1)).Evenso,intheexperimentalresults(Table2)we observeamuchhigherincreaseintheretentiontimes,namelyfrom 67%(forChannelIandacrossflowrateof0.8mL/min)to180%(for ChannelIIandacross-flowrateof1.0mL/min)forthemonomerof apoferritin,whichindicatesthattheeffectofthegrooveshasthe largestcontributiontotheincreaseintheretentiontime.Moreover, overloadingwasinvestigatedbyinjectingdifferentsamplemass, namely2g,10g,and20g,intheChannelI/MSmembraneI system;nooverloadingeffectwasobservedasretentiontime,plate heightandselectivitywerepracticallythesameforeveryexamined injectedmass
4.3 Computationalfluiddynamics FortheCFDexperiments,theminiaturized channel(Channel
IIin Fig 4)wasmodelled and themigration ofthe apoferritin monomeranddimerwassimulated.Thediffusioncoefficientsfor themonomerandfor thedimer ofapoferritinweretakenfrom theAF4experiments(wheretheratioofthediffusioncoefficients, andtherefore theselectivitywas1.34).Themodelwasverified
byreducingsignificantlythesizeofthemeshelements,thetime
Trang 9Table 3
CFD experiments and comparison with the experimental results for apoferritin,
Channel II and cross-flow rate 0.5 mL/min.
stepandthetolerances;allthesechangesdidnotalterthe
reten-tiontimes(within0.2%).TheCFDmodelwasvalidatedcomparing
theresults(retentiontimeandselectivity)withtheexperimental
resultsobtainedforthenonpatternedmembrane.Goodagreement
wasfound,thestandarderror(SE)was2%fortheretentiontime
ofapoferritin (monomeror dimer)and<1%for theirselectivity
(Table3).Theassumptionofthelaminarflowconditions,which
wasusedforthemodel,wasjustifiedbytheresults;theReynolds
numberwaslessthan0.05acrossthewholechannel(maximum
inthemiddleofthechannelthickness)andlessthan2·10−4inside
thegrooves
For the patterned membrane (MS membrane II) and
˙Vc=0.5mL/min, the flow and concentration profiles, and the
derived concentration at the outlet for each time point are
depictedinFig.6.Itwasrevealedthattheexperimentalretention
time and selectivity are much lower compared to the values
predicted by the simulation (Table 3) This may be due to a
non-uniform cross-flow velocity as a result of differences in
themembranecompactionand/orintheporesizebetweenthe
ridgesandthecavitiesofthemembrane’sselectivelayerasitwas
discussedabove
5 Conclusions
Todateonlyflat(non-patterned)UFmembraneshavebeenused
inAF4asmicron-sizedfeaturesareconsideredharmfulforthe
sep-aration.Wehavedemonstratedthatmicron-sizedgroovescouldin
factimproveperformanceinAF4.Thiswasshownbyseveralmeans
includingmomentanalysis,physicalexperiments,andCFD
simu-lations.Ourresultsshowthatperpendiculargroovescanincrease
retention,selectivityandresolution.Thissystemcouldbeusefulas
macromoleculesandnanoparticlescouldbeanalyzedwithlower
cross-flowrateswithouttheneedtousehigherspacerthickness
orhigher cross-flowtooutletflow ratio.Thisconcept couldbe
appliedonanyFFFsystemasithasbeenoriginallydemonstrated
byGiddingsetal.forThFFF[12]
Thephysical experimentswerecarried outwith
microstruc-turedUFmembranesfabricatedbyhot-embossing.Thisfabrication
processcausedanincreaseintheactualMWCOofthemembrane
(asindicatedbytheAF4experiments)buttheeffectofthegrooves
couldbeshownwiththelargerproteinstandardsusedinthisstudy
(apoferritin 443kDa and thyroglobulin 669kDa) However, this
conceptcouldbeparticularlyusefulforsmallermacromolecules,
andthereforefutureworkshouldbefocusedonthefabricationof
microstructuredmembraneswithlowerMWCOandhighwater
permeability This couldbe achieved,for instance, by methods
otherthanhotembossingsuchasphaseseparationoradditive
tech-nologies(e.g.,3Dprintingofanon-porousorporousmaterialover
anUFmembrane).AdditionalresearchwithCFDexperimentsof
differentgrooveshapesanddimensionsisunderwaytoinvestigate
theoptimalgroovestructure
Acknowledgements
Thisworkwas partof theresearchprogram SmartSep with
projectnumber 11400 which wasfinanced by theNetherlands
OrganizationforScientificResearch(NWO).Authorsalso acknowl-edgeLydiaBolhuis-Versteeg(UniversityofTwente)forherhelpon SEMimagingandWyattTechnologyEuropeforprovidingtechnical assistance
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