45 Ti 转子用户手册 conv

L5-TB-036VAFebruary2014

Type45TiRotor

UsedInBeckmanCoulterClassH,R,andSPreparativeUltracentrifuges

©2014BeckmanCoulter,Inc.

Type45TiRotor

Trademarks

BeckmanCoulter,thestylizedlogo,andQuick-SealaretrademarksofBeckmanCoulter,Inc.andareregisteredintheUSPTO.

Allothertrademarks,servicemarks,products,orservicesaretrade-marksorregisteredtrademarksoftheirrespectiveholders.

2

Type45TiRotor

! SAFETYNOTICE

Thissafetynoticesummarizesinformationbasictothesafeuseoftherotordescribedinthismanual.Theinternationalsymboldisplayedaboveisaremindertotheuserthatallsafetyinstructionsshouldbereadandunderstoodbeforeoperationormaintenanceofthisequipmentisattempted.Whenyouseethesymbolonotherpagesthroughoutthispublication,payspecialattentiontothespecificsafetyinformationpresented.Observanceofsafetyprecautionswillalsohelptoavoidactionsthatcoulddamageoradverselyaffecttheperformanceoftherotor.Thisrotorwasdeveloped,manufactured,andtestedforsafetyandreliabilityaspartofaBeckmanCoulterultracentrifuge/rotorsystem.ItssafetyorreliabilitycannotbeassuredifusedinacentrifugenotofBeckmanCoulter’smanufactureorinaBeckmanCoulterultracentri-fugethathasbeenmodifiedwithoutBeckmanCoulter’sapproval.

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Handlebodyfluidswithcarebecausetheycantransmitdisease.Noknowntestofferscompleteassurancethatsuchfluidsarefreeofmicro-organisms.Someofthemostvirulent—Hepatitis(BandC)viruses,HIV(I–V),atypicalmycobacteria,andcertainsystemicfungi—furtheremphasizetheneedforaerosolprotection.

Handleotherinfectioussamplesaccordingtogoodlaboratoryproceduresandmethodstopreventspreadofdisease.Becausespillsmaygenerateaerosols,observepropersafetyprecautionsforaerosolcontainment.Donotruntoxic,pathogenic,orradioactivematerialsinthiscentrifugewithouttakingappropriatesafetyprecautions.BiosafecontainmentshouldbeusedwhenRiskGroupIImaterials(asidentifiedintheWorldHealthOrganizationLaboratoryBiosafetyManual)arehandled;materialsofahighergrouprequiremorethanonelevelofprotection.

Therotorandaccessoriesarenotdesignedforusewithmaterialscapableofdevelopingflammableorexplosivevapors.Donotcentrifugesuchmaterialsinnorhandleorstorethemneartheultracentrifuge.

AlthoughrotorcomponentsandaccessoriesmadebyothermanufacturersmayfitintheType45Tirotor,theirsafetyinthisrotorcannotbeascertainedbyBeckmanCoulter.Useofothermanufacturers’componentsoraccessoriesintheType45Tirotormayvoidtherotor

warrantyandshouldbeprohibitedbyyourlaboratorysafetyofficer.Onlythecomponentsandaccessorieslistedinthispublicationshouldbeusedinthisrotor.

Makesurethatfilledcontainersareloadedsymmetricallyintotherotorandthatopposingtubesarefilledtothesamelevelwithliquidofthesamedensity.Makesurethatcavitiesinusehavetheproperspacersinserted(ifapplicable)beforeinstallingtherotorlid.

Ifdisassemblyrevealsevidenceofleakage,youshouldassumethatsomefluidescapedtherotor.Applyappropriatedecontaminationprocedurestothecentrifugeandaccessories.

Neverexceedthemaximumratedspeedoftherotorandlabwareinuse.RefertothesectiononRUNSPEEDS,andderatetherunspeedasappropriate.

Donotusesharptoolsontherotorthatcouldcausescratchesintherotorsurface.Corrosionbeginsinscratchesandmayopenfissuresintherotorwithcontinueduse.

3

Type45TiRotor

TYPE45TiROTOR

24°

rmin

rav

rmax

AxisofRotation

SPECIFICATIONS

Maximumspeed....................................45000rpmDensityratingatmaximumspeed.........................1.2g/mLRelativeCentrifugalField*atmaximumspeed

Atrmax(103.8mm).............................235000×gAtrav(69.8mm)................................158000×gAtrmin(35.9mm)................................81400×g

kfactoratmaximumspeed..................................133Conditionsrequiringspeedreductions...............seeRUNSPEEDSNumberoftubecavities......................................6Availabletubes......................................seeTable1Nominaltubedimensions(largesttube).................38×102mmNominaltubecapacity(largesttube)........................94mLNominalrotorcapacity..................................564mLApproximateaccelerationtimetomaximumspeed

(fullyloaded)........................................9minApproximatedecelerationtimefrommaximumspeed

(fullyloaded).....................................91/2minWeightoffullyloadedrotor..........................12kg(26lb)Rotormaterial.........................................titanium

rω

2

g

RPM

1000

⎛ ⎞

*RelativeCentrifugalField(RCF)istheratioofthecentrifugalaccelerationataspecifiedradius

andspeed(rω2)tothestandardaccelerationofgravity(g)accordingtothefollowingformula:

RCF=

whereristheradiusinmillimeters,ωistheangularvelocityinradianspersecond(2πRPM/60),andgisthestandardaccelerationofgravity(9807mm/s2).Aftersubstitution:

RCF=1.12r⎝⎠2

4

Type45TiRotor

DESCRIPTION

ThisrotorhasbeenmanufacturedinaregisteredISO9001or13485facilityforusewiththeappropriatelyclassifiedBeckmanCoulterultracentrifuge.

Handle(339164)

Lid(339163)

LidO-ring

(878260)

HandleO-ring(854519)

RotorBody

OverspeedDisk(335458)

TheType45Ti,ratedfor45000rpm,isafixedanglerotordesignedtocentrifugeuptosixtubesata24-degreeangletotheaxisofrotation.UsedinBeckmanCoulterclassH,R,andSpreparativeultracentrifuges,therotordevelopscentrifugalforcessufficientforapplicationsincludingpelletingofsmallparticles,andpurificationofvirusesandsubcellularorganellesusingsucrosegradients.Upto564mLofsampleandgradientcanbecentrifugedperrun.

Therotorismadeoftitaniumandisfinishedwithblackpolyurethanepaint(aslightlyrough,orgrainy,surfaceisnormal).Thealuminumlidandhandlearered-anodizedforcorrosionresistance.Thecoloralsoidentifiestherotorasahigh-performancerotorthatusesredorbluealuminumcapswithmaximum-diameterplastictubes.O-ringsmadeofBunaNrubberinthelidandhandlemaintainatmosphericpressureinsidetherotorduringcentrifugation,iftheyareproperlylubricated.Foursmallholesinthelidprovideatemporaryvent,preventingpressurebuild-upandextrusionofthelargelidO-ring

intheeventofleakage.Becauseoftheweightoftherotor,drivepinsarenotrequiredintherotordrivehubcavity.

Therotorisequippedwithanoverspeeddiskontherotorbottom.Rotorspeedexceeding45000rpmwillbedetectedininstrumentswiththephotoelectricoverspeeddetectionsystem.

SeetheWarrantyatthebackofthismanualforwarrantyinformation.

PREPARATIONANDUSE

SpecificinformationabouttheType45Tirotorisgivenhere.Infor-mationcommontothisandotherrotorsiscontainedinRotorsandTubesforPreparativeUltracentrifuges(publicationLR-IM)whichaccompaniesthismanual.Usethisrotormanual,togetherwithpublicationLR-IM,forcompleterotorandaccessoryoperation.

5

Type45TiRotor

NOTE

AlthoughrotorcomponentsandaccessoriesmadebyothermanufacturersmayfitintheType45Tirotor,theirsafetyinthisrotorcannotbeascertainedbyBeckmanCoulter.Useofothermanufacturers’componentsoraccessoriesintheType45Tirotormayvoidtherotorwarrantyandshouldbeprohibitedbyyourlaboratorysafetyofficer.Onlythecomponentsandacces-sorieslistedinthispublicationshouldbeusedinthisrotor.

PRERUNSAFETYCHECKS

ReadtheSafetyNoticepageatthefrontofthismanualbeforeusingtherotor.

45000-rpm40-Sector(335458)

1.Makesurethattherotorandlidarecleanandshownosignsofcorrosionorcracking.

2.Makesurethatthe40-sector(45000rpm)overspeeddiskisproperlyattachedtotherotorbottom.Ifthediskismissingordamaged,replaceitaccordingtotheinstructionsinRotorsandTubes.

3.Checkthechemicalcompatibilitiesofallmaterialsused(refertoAppendixAinRotorsandTubes).

4.VerifythatthetubesandbottlesbeingusedarelistedinTable1.

ROTORPREPARATION

Forrunsatotherthanroomtemperature,refrigerateorwarmtherotorbeforehandforfastequilibration.

1.BesurethatmetalthreadsintherotorarecleanandlightlybutevenlylubricatedwithSpinkotelubricant(306812).AlsoensurethatO-ringsarelightlybutevenlycoatedwithsiliconevacuumgrease(335148).

6

Type45TiRotor

2.Loadthefilledcontainerssymmetricallyintotherotor.(RefertoTUBESANDBOTTLESonpage8forinformationaboutcontainers.)Iffewerthansixtubesarebeingrun,theymustbe

arrangedsymmetricallyintherotor(seeFigure1).Opposingtubesmustbefilledtothesamelevelwithliquidofthesamedensity.

Figure1.ArrangingTubesintheRotor.Two,three,four,orsixtubescanbecentrifugedperruniftheyarearrangedintherotorasshown.

3.Completeloadingbyplacingthecorrectspacers(ifrequired)overthetubes.

NOTE

Placefilledtubesinatleasttwoopposingcavities.Makesurethatcavitiesinusehavetheproperspacersinserted(ifapplicable)beforeinstallingtherotorlid.

4.Putthelidinplaceandtightenbyhandasfirmlyaspossible.

7

Type45TiRotor

OPERATION

Lowertherotorstraightdownontothedrivehub.

1.Carefullyplacetherotoronthedrivehub.

2.Refertotheapplicableinstrumentinstructionmanualforultracen-trifugeoperation.

3.Foradditionaloperatinginformation,seethefollowing:

•RUNTIMES,page13,forusingkfactorstoadjustrundurations

•RUNSPEEDS,page14,forinformationaboutspeedlimitations

•SELECTINGCsClGRADIENTS,page16,formethodstoavoidCsClprecipitationduringcentrifugation

REMOVALANDSAMPLERECOVERY

!CAUTION

Ifdisassemblyrevealsevidenceofleakage,youshouldassumethatsomefluidescapedtherotor.Applyappropriatedecontaminationprocedurestothecentrifugeandaccessories.

1.Removetherotorfromthecentrifugebyliftingitstraightupandoffthedrivehub.

Quick-SealTubeRemovalTool(361668)

2.Removetherotorlid.

3.Usetheappropriateremovaltool(listedintheSUPPLYLIST)toremovethespacersandtubes.

TUBESANDBOTTLES

TheType45TirotorusesthetubesandbottleslistedinTable1.Besuretouseonlythoseitemslisted,andtoobservethemaximumspeedlimitsandfillvolumesshown.(Maximumfillvolumeisthemaximumamountthatcanbecentrifugedinthecontainerlisted.)

RefertoAppendixAinRotorsandTubesforchemicalcompatibili-tiesoftube,bottle,andaccessorymaterials.

8

Type45TiRotor

Table1.AvailableTubesandBottlesfortheType45TiRotor.Useonlytheitemslistedhere.

Tube RequiredAccessory

Dimensions

38×102mm

38×102mm

38×102mm

38×102mm

38×102mm

Description

Quick-SealUltra-Clear

Quick-Sealpolypropylene

Ultra-Clear

thinwallpolypropylene

thickwallpolypropylene

PartNumber

345778(pkg/25)

345776(pkg/25)

345777(pkg/25)

345775(pkg/25)

355643(pkg/25)

MaxFillVol

94mL

94mL

94mL

94mL

81mL

Description

titaniumspacer

titaniumspacer

redaluminumcap

redaluminumcap

bluealuminumcap

PartNumber

342697

342697

330901a

330901b

338905

MaxSpeed/RCF/

kFactor

45000rpm235000×g133

45000rpm235000×g133

45000rpm235000×g133

45000rpm235000×g133

45000rpm235000×g133

47mLnone—15000rpm26200×g

1195

38×102mm thickwallpolycarbonate

355628 81mL bluealuminumcap 338905(pkg/25)

45000rpm235000×g25

47mLnone—15000rpm26200×g

1195

38×102mm

16×76mm

16×76mm

16×76mm

polycarbonatebottleassembly(bottleonly—355655)

Ultra-Clear

thinwallpolypropylene

thickwallpolypropylene

355622(pkg/6)

344085(pkg/50)

326814(pkg/50)

355640(pkg/25)

70mL aluminumcap

13.5mL Delrindadaptere

aluminumcap

13.5mL Delrinadaptere

aluminumcap

13.5mL Delrinadaptere

aluminumcapf

35562345000rpmc235000×g

133

30344839000rpm149000×g

330860 107

30344839000rpm149000×g

330860 107

30344839000rpm149000×g

338907 107

—continued

9

Type45TiRotor

Table1.AvailableTubesandBottlesfortheType45TiRotor(continued)

Tube RequiredAccessory

Dimensions

16×76mm

13×89mm

13×89mm

13×89mm

13×89mm

13×64mm

13×64mm

13×64mm

13×64mm

13×51mm

13×64mm

Description

thickwallpolycarbonate

Ultra-Clear

thinwallpolypropylene

thickwallpolypropylene

thickwallpolycarbonate

Ultra-Clear

thinwallpolypropylene

thickwallpolypropylene

thickwallpolycarbonate

thickwallpolypropylene

thickwallpolycarbonate

PartNumber

355630(pkg/25)

344087(pkg/50)

326822(pkg/50)

355639(pkg/25)

355629(pkg/25)

344088(pkg/50)

326820(pkg/50)

355644(pkg/25)

355645(pkg/25)

355641(pkg/25)

355632(pkg/25)

MaxFillVol

13.5mL

10.5mL

10.5mL

10.5mL

10.5mL

6.5mL

6.5mL

4mL

4mL

3.5mLi

3.5mLi

Description

Delrinadaptere

aluminumcapg

Delrinadaptere

aluminumcap

Delrinadaptere

aluminumcap

Delrinadaptere

Delrinadaptere

Delrinadaptere

aluminumcap

Delrinadaptere

aluminumcap

Delrinadaptere

Delrinadaptere

aluminumadapter(holds7tubes)

aluminumadapter(holds7tubes)

PartNumber

303448

338907

303459

346256h

303459

346256h

303459

303459

303449

346256h

303449

346256g

303449

303449

350781

350781

MaxSpeed/RCF/

kFactor

39000rpm149000×g107

39000rpm156000×g110

39000rpm156000×g110

39000rpm156000×g110

39000rpm156000×g110

39000rpm138000×g90

39000rpm138000×g90

39000rpm138000×g90

39000rpm138000×g90

30000rpm91000×g151j

30000rpm91000×g151j

aUseonlyneopreneO-ring(346242).bUseneopreneO-ring(346242or885511).

cForlowerfillvolumes(notlessthan15mL)reducemaximumspeedto35000rpm.dDelrinisaregisteredtrademarkofE.I.DuPontdeNemours&Company.

eEachadapterholdsonetube.

fCapsareoptionalwiththesetubes;caplessfillvolumeis8mL.Whenrunwithoutcaps,reducespeedto15000rpm.gCapsareoptionalwiththesetubes;caplessfillvolumeis8mL.Whenrunwithoutcaps,reducespeedto30000rpm.hCapassemblyincludesneoprenegasket(344672)andstem(346246).

iFillto13mmfromthetubetop.

jMaximumRCFandkfactoraremeasuredatthecentralcavityoftheseven-placeadapter.

10

Type45TiRotor

TemperatureLimits

•Plastictubesandbottleshavebeencentrifugetestedforuseattemperaturesbetween2and25°C.Forcentrifugationatothertemperatures,pretesttubesunderanticipatedrunconditions.

•Ifplasticcontainersarefrozenbeforeuse,makesurethattheyarethawedtoatleast2°Cpriortocentrifugation.

Quick-SealTubes

Quick-Sealtubesmustbesealedpriortocentrifugation.Thesetubesareheatsealedanddonotneedcaps;however,spacersarerequiredontopofthetubeswhentheyareloadedintotherotor.

•FillQuick-Sealtubesleavingasmallbubbleofairatthebaseoftheneck.Donotleavealargeairspace—toomuchaircancauseexcessivetubedeformation.

•RefertoRotorsandTubes,fordetailedinformationontheuseandcareofQuick-Sealtubes.

ThinwallTubes

ThinwallpolypropyleneandUltra-Cleartubesrequirecapsfortubesupport.Fillthetubesasfullaspossibletopreventtubecollapseduringcentrifugation.Ifnecessary,floatmineraloil(orsomeotherlow-density,immiscibleliquid)ontopofthetubecontentstofillthetubetoitsmaximumvolume.(DonotuseanoiloverlayinUltra-Cleartubes.)

ThickwallTubes

Thickwallpolypropyleneandpolycarbonatetubescanberunpartiallyfilled(atleasthalffilled)withorwithoutcaps,butallopposingtubesforarunmustbefilledtothesamelevelwithliquidofthesamedensity.Donotoverfillcaplesstubes;besuretonotethereductionsinfillvolumeandrunspeedshowninTable1.

11

Type45TiRotor

TubeCaps

O-RingorGasket

Thinwalltubesrequirecapsfortubesupportduringcentrifugation;thickwalltubesandbottlescanberunwithorwithoutcaps(speedreductionmayberequired).UseonlythecapslistedinTable1.RefertoRotorsandTubesfortheuse,assembly,andmaintenanceofcaps.

•InspecttubecapsbeforeuseasdescribedinRotorsandTubes.Replaceanydamagedcomponents.

•InspecttheO-ringsorgasketsinthecapsforcracks,nicks,orflattenedareas.Besurethattheyaredryandfreeoflubricantduringassembly.

Inserttubefrombottom

Tube-CapVise(305075)

•Tightenaluminumcapswithahexdriverwhilethetubeisheldinthetube-capvise(305075).

•Tightenbottlecapsbyhand.

NOTE

Thinwalltubeswithred-orblue-anodizedcapsmustbecapped.Thehigh-strengthaluminumcapslistedhavebeenspeciallydesignedforuse

withthe38-mmdiameterthinwallUltra-Clearandpolypropylenetubes;donotuseanyothercapswiththesetubes.RefertoInstructionsforUsingAluminumTubeCaps(publication

L5-TB-060)fortheuse,assembly,andmainte-nanceofthesecaps.Tightenthecapswithatorquewrenchto11N•m(100in.-lb).

PolycarbonateBottles

Thecappedpolycarbonatebottlesmaybecentrifugedcompletelyfilled,orpartiallyfilled(notlessthanhalffull).Again,allopposingcontainersforarunmustbefilledtothesamelevel.BesuretonotethereductionsinrunspeedshowninTable1ifbottlesarepartiallyfilled.

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Type45TiRotor

MultitubeAdapters

Index Seven-placealuminumadapters(350781)areavailabletoenableMark runningsmall-volumethickwalltubesinthisrotor.Iffewerthansix

oftheseadaptersarerun,loadtherotorsymmetrically.Orienttheindexmarkonthetopsurfaceofeachadaptertowardtheoutsideoftherotor.

NOTE

Setsofadaptersarematchedbyweight.Donotintermixadaptersfromdifferentsetsunlessyouhaveweighedthemandalladaptersforarunweighthesametowithin0.1gram.

RUNTIMES

TIMEHR:MIN

Thekfactoroftherotorisameasureoftherotor’spelletingeffi-ciency.(BeckmanCoulterhascalculatedthekfactorsforallofitspreparativerotorsatmaximumratedspeedandusingfulltubes.)Thekfactoriscalculatedfromtheformula:

ln(r ⁄r ) 13

min

10

3600

max

2

k=×

ω

(1)

whereωistheangularvelocityoftherotorinradianspersecond(ω=0.105×rpm),rmaxisthemaximumradius,andrministheminimumradius.

Aftersubstitution:

(2.533×1011)ln(r ⁄r )

max

min

2

k=

rpm

(2)

Usethekfactorinthefollowingequationtoestimatetheruntimet(inhours)requiredtopelletparticlesofknownsedimentationcoeffi-cients(inSvedbergunits,S).

k

s

t=-- (3)

13

Type45TiRotor

Runtimescanbeestimatedforcentrifugationatlessthanmaximumspeedbyadjustingthekfactorasfollows:

45000

actualrunspeed

⎛ ⎞

kadj=k⎝⎠2 (4)

Runtimescanalsobeestimatedfromdataestablishedinpriorexperi-mentsusingadifferentrotorifthekfactorofthepreviousrotorisknown.Foranytworotors,aandb:

a a

t k

-t=-k (5)b b

FormoreinformationonkfactorsseepublicationDS-719,Useof

kFactorforEstimatingRunTimesfromPreviouslyEstablishedRunConditions.

RUNSPEEDS

SPEEDRPM/RCF Thecentrifugalforceatagivenradiusinarotorisafunctionofspeed.Comparisonsofforcesbetweendifferentrotorsaremadebycomparingtherotors’relativecentrifugalfields(RCF).When

rotationalspeedisadjustedsothatidenticalsamplesaresubjectedtothesameRCFintwodifferentrotors,thesamplesaresubjectedtothesameforce.TheRCFatanumberofrotorspeedsisprovidedinTable2.

DonotselectrotationalspeedsthatexceedthelimitsinTable1.Inaddition,speedsmustbereducedunderthefollowingcircumstances:

1.Ifnonprecipitatingsolutionsmoredensethan1.2g/mLarecentrifuged,themaximumallowablerunspeedmustbereducedaccordingtothefollowingequation:

1.2g/mL

ρ

reducedmaximumspeed=(45000rpm) (6)

whereρisthedensityoftubecontents.Thisspeedreductionwillprotecttherotorfromexcessivestressesduetotheaddedtubeload.Note,however,thattheuseofthisformulamaystillproducemaximumspeedsthatarehigherthanthelimitationsimposedbytheuseofcertaintubesoradapters(seeTable1).Insuchcases,usethelowerofthetwospeeds.

14

Type45TiRotorRCF(xg)

Table2.RelativeCentrifugalFieldsfortheType45TiRotor.EntriesinthistablearecalculatedfromtheformulaRCF=1.12r(RPM/1000)2

andthenroundedtothreesignificantdigits.

RelativeCentrifugalField(xg)

Rotor

Speed Atrmax(rpm)(103.8mm)

45000 23500041000 19500040000 186000

350001050002500072700

20000465001500026200

10000 11600

Atrav(69.8mm)

158000131000125000

958007040048900

3130017600

7810

Atrmin(35.9mm)

814006760064000

493003620025100

161009050

4010

kFactor*

133160168

220299430

6721195

2689

*CalculatedforallBeckmanCoulterpreparativerotorsasameasureoftherotor’srelativepelletingefficiencyinwaterat20°C.

.

RelativeCentrifugalFieldsType45TiRotor

250000

200000

150000 rmaxrav

100000 rmin

50000

0

0 5000 10000 15000 20000 25000 30000 35000 40000 45000

Speed(rpm)

15

Type45TiRotor

2.FurtherspeedlimitsmustbeimposedwhenCsClorotherself-forming-gradientsaltsarecentrifuged,asequation(6)doesnotpredictconcentrationlimits/speedsthatarerequiredtoavoidprecipitationofsaltcrystals.SolidCsClhasadensityof4g/mL,andifprecipitatedduringcentrifugationmaycausecatastrophicrotorfailureandinstrumentdamage.Figures2and3,togetherwiththedescriptionandexamplesbelow,showhowtoreducerunspeedswhenusingCsClgradients.

SELECTINGCsClGRADIENTS

Precipitationduringcentrifugationwouldalterdensitydistribution,andthiswouldchangethepositionofthesamplebands.CurvesinFigures2and3areprovideduptothemaximumratedspeedoftherotor,butnotealsothattubesorbottlesmustneverbecentrifugedfasterthanthelimitsinTable1.

NOTE

ThecurvesinFigures2and3areforsolutionsofCsClsaltdissolvedindistilledwateronly.Ifothersaltsarepresentinsignificantconcentra-tions,theoverallCsClconcentrationmayneedtobereduced.

RotorspeedisusedtocontroltheslopeofaCsCldensitygradient,andmustbelimitedsothatCsClprecipitationisavoided.SpeedanddensitycombinationsthatintersectonorbelowthecurvesinFigure3ensurethatCsClwillnotprecipitateduringcentrifugationintheType45Tirotor.Curvesareprovidedattwotemperatures:20°C(blackcurves)and4°C(graycurves).

ThereferencecurvesinFigure3showgradientdistributionatequi-librium.EachcurveinFigure3iswithinthedensitylimitsallowedfortheType45Tirotor:eachcurvewasgeneratedforasinglerunspeedusingthemaximumallowablehomogeneousCsCldensities(oneforeachfilllevel)thatavoidprecipitationatthatspeed.(ThegradientsinFigure3canbegeneratedfromsteporlineargradients,orfromhomogeneoussolutions.ButthetotalamountofCsClinsolutionmustbeequivalenttoahomogeneoussolutioncorrespondingtotheconcentrationsspecifiedinFigure3.)Figure3canalsobeusedtoapproximatethebandingpositionsofsampleparticles.

16

Type45TiRotor

3/4

3/4

full

full

1/4

1/4

1/2

1/2

1.90

full

3/4

1/2 1/4

1.80

1.70

1.60

HomogeneousCsClSolution(g/mL)

1.50

1.40

1.30

TYPE45TiROTOR

1.20

1.10

0 10 20 30 40 45RotorSpeed(Krpm)

Figure2.PrecipitationCurvesfortheType45TiRotor.UsingcombinationsofrotorspeedsandhomogeneousCsClsolutiondensitiesthatintersectonorbelowthesecurvesensuresthatCsClwillnotprecipitateduringcentrifugation.Thedashedlineisarepresentationofequation(6),andisshownheretoillustratetheinabilityofthatequationtopredictCsClprecipitation.

17

Type45TiRotor

25000rpm

25000rpm

30000rpm

30000rpm

35000rpm

35000rpm

41000rpm

41000rpm

45000rpm

45000rpm

1.90

TYPE45TIROTOR

Eachsquareonthegrid

represents1.27mmby

1.80 0.01g/mL.

1.70

1.60

Density(g/mL)

1.50

1.40

1.30

1.20

1.10

1.00

35.9 53.3rmin3/4-filled

69.81/2-filled

86.4103.81/4-filledrmax

DistancefromAxisofRotation(mm)

Figure3.CsClGradientsatEquilibriumfortheType45TiRotor.CentrifugationofhomogeneousCsClsolutionsatthemaximumallowablespeeds(fromFigure2)resultsingradientspresentedhere.

18

Type45TiRotor

ADJUSTINGFILLVOLUMES

Buffer

Gradient

Figures2and3showthatseveralfillvolumesarepossibleinatube.Ifatubeispartiallyfilledwithgradientsolution,floatmineraloil(orsomeotherlow-density,immiscibleliquid)ontopofthetubecontentstofillthetubetoitsmaximumvolume.(DonotuseanoiloverlayinUltra-Cleartubes.)NotethatforagivenCsCldensity,

asthefillleveldecreasesthemaximumallowablespeedincreases.Partialfillingmaybedesirablewhenthereislittlesampleorwhenyouwishtoshortentheruntime.

Forexample,aquarter-filledtubeof1.55-g/mLhomogeneousCsClsolutionat20°Cmaybecentrifugedat45000rpm(seeFigure2).Thesegmentofthe45000-rpmcurve(Figure3)fromthequarter-filledlineto1.86g/mLatthetubebottomrepresentsthisgradient.Thesamesolutioninahalf-filledtubemaybecentrifugednofasterthan42500rpm,and41000rpminathree-quarter-filledtube.(GradientcurvesnotshowninFigure3canbeinterpolated.)Atubefullof

the1.55-g/mLCsClsolutionmaybecentrifugednofasterthan35000rpm.(Notethatthe45000-rpmcurvesinFigure3areidenticalfor4and20°C.)

TYPICALEXAMPLESFORDETERMININGCsClRUNPARAMETERS

AtSpeed

FloatingComponents

ExampleA:Knowinghomogeneous