Appendix F: Centrifugation

FigureF.1.CrosssectionofSorvallSS-34rotor.

Rotorsforacentrifugeareeitherfixedangles,swingingbuckets,continuousflow,orzonal,dependinguponwhetherthesampleisheldatagivenangletotherotationplane,isallowedtoswingoutonapivotandintotheplaneofrotation,designedwithinletandoutletportsforseparationoflargevolumes,oracombinationofthese.FigureF.1demonstratesthecharacteristicsofeachofthese.

Fixedanglesgenerallyworkfaster;substancesprecipitatefasterinagivenrotationalenvironment,ortheyhaveanincreasedrelativecentrifugalforceforagivenrotorspeedandrADIus.Theyalsohavefew(orno)movingpartsontherotoritselfandthushavevirtuallynomajormechanicalfailures,otherthanpotentialmetalstress,whichallrotorsundergo.Theserotorsarethework-horseelementsofacelllaboratory,andthemostcommonisarotorholding8centrifugetubesatanangleof34°Cfromthevertical(suchastheSorvallSS-34rotorortheBeckmanJA-20).FigureF.1presentsacross-sectionaldiagramoftheSorvallSS-34.

Swingingbucketrotors(alsoknownashorizontalrotors)havetheadvantagethatthereisusuallyacleanmeniscusofminimumarea.Inafixedanglerotor,thematerialsareforcedagainstthesideofthecentrifugetube,andthenslidedownthewallofthetube.Thisactionistheprimaryreasonfortheirapparentfasterseparation,butalsoleadstoabrasionoftheparticlesalongthewallofthecentrifugetube.Foraswingingbucket,thematerialsmusttraveldowntheentirelengthofthecentrifugetubeandalwaysthroughthemediawithinthetube.Sincethemediaisusuallyaviscoussubstance,theswingingbucketappearstohavealowerrelativecentrifugalforce,thatisittakeslongertoprecipitateanythingcontainedwithin.If,however,thepointofcentrifugationistoseparatemoleculesororganellesonthebasisoftheirmovementsthroughaviscousfield,thentheswingingbucketistherotorofchoice.Moreover,ifthereisadangerorscrapingoffanoutershellofaparticle(suchastheoutermembraneofachloroplast),thentheswingingbucketistherotorofchoice.Mostcommonclinicalcentrifuges1haveswingingbuckets.Sincethebucketsareeasytointerchange,thistypeofrotorisextremelyversatile.Itsmajordrawbackisthenumberofmovingpartswhicharepronetofailurewithextendeduse.

Nearlyallcellbiologylaboratorieswillhaveseveralexamplesoffixedangleandhorizontalrotors.Whilethesamplevolumesoftheserotorscanbesignificant,theyarelimiting.Toovercomethislimitation,acontinuousflowcentrifugecanbeused.Limnologistsoftenemploysuchadevicetoseparateplanktonfromgallonsoflakewater.Cellbiologistsemployzonalrotorsforthelargescaleseparationofparticlesondensitygradients.Zonalrotorscancontainupto2litersofsolutionandcanworkwithtissuesamplesmeasuredinounces(orevenpounds).Therotorsarebroughtuptoabout3000RPMwhileempty,andthedensitymediaandtissuesareaddedthroughspecializedports.Thistypeofrotorhasadistinctpreparativeadvantageoverthegradientcapacityofmoretypicalrotors.

ROTORTUBES

Forbiologicalwork,thetubesaredividedintofunctionalgroups,madeofregularglass,Corexglass,nitrocellulose,orpolyallomer.Regularglasscentrifugetubescanbeusedatspeedsbelow3,000RPM,thatisinastandardclinicalcentrifuge.Abovethisspeed,thexgforceswillshattertheglass.

AspecialhighspeedglasswiththetradenameofCorex(CorningGlassWorks)hasbeendevelopedtohandlespeedsupto15-18,000RPM.Thesetubescanbeusedinmostroutinecellorganellepreparations,if,andonlyif,theproperadaptersarealsousedwithinthecentrifugerotors.Thesetubesarerelativelyexpensive(about$3.50each)andshouldneverbeusedforanypurposeotherthanthecentrifuge.Anytubeswithscratchesorchipsshouldbedisposedofimmediately.Thesehigh-speedglasstubeswillshatterabove18,000RPM.

Forworkinthehigherspeedranges,centrifugetubesaremadeofplasticornitrocellulose.Preparativecentrifugetubesaremadeofpolypropylene(sometimespolyethylene)andcanwithstandspeedsupto20,000RPM.Thesetubesshouldbecarefullyexaminedforstressfracturesbeforeuse.Atubewithafracturewillholdfluidsbeforecentrifugation,butthecrackswillopenundercentrifugalforce.

Nitrocelluloseareinexpensiveandusedformostultracentrifugation.Theyaremeanttobeusedonlyonceandthendiscarded.Repeateduseincreasesthechanceoftubecollapseduetointernalmolecularstresswithinthetubewalls.Thereisnowaytopre-determinethis,soitisbesttoalwaysuseanewtubeforultracentrifugation.Nitrocellulosealsobecomeslessflexiblewithage,andthepurchasedateforalltubesshouldbenoted.Tubesolderthan1yearshouldbediscarded.Acentrifugetubeisinexpensivewhencomparedtothelossoftimeandmaterialsforatypicalultracentrifugerun.

Polyallomertubesarere-usable,moreexpensive,andslippery.Moleculeswillslidedownthewallsofthesetubesmoreeasily,andthusarethetubesofchoiceforprecipitationcentrifugations.Theyarealsomorechemicallyinert.

RELATIVECENTRIFUGALFORCE

FigureF.2.NomogramforR.C.F.

Moderndayultracentrifugescangenerateforcesinexcessof300,000timesthatofgravity,forcessufficienttoovercometheverycohesionofmostmolecules(includingthemetaloftherotor).Theforceisusuallygivenassomevaluetimesthatofgravity.

Thecentrifugalforceisdependentupontheradiusoftherotationoftherotor,thespeedatwhichitrotates,andthedesignoftherotoritself(fixedangle,vsswingingbucket).Rotorspeedanddesigncanbeheldconstant,buttheradiuswillvaryfromthetopofacentrifugetubetothebottom.Ifameasurementfortheradiusistakenasthemid-point,orasanaverageradius,andallforcesaremathematicallyrelatedtogravity,thenoneobtainsarelativecentrifugalforce,labeledasxg.Centrifugationproceduresaregivenasxgmeasures,sinceRPMandotherparameterswillvarywiththeparticularinstrumentandrotorused.RelativeCentrifugalForceisaconstantthatisindependentoftheapparatusused.

FigureF.2presentsaNomogramforcalculationofR.C.F.foragivenradiusandRPM.Asimpleformulaforcalculatingthisvalueis:

RCF=1.12r(RPM/1000)wherer=radiusinmillimetersRPM=revolutionsperminute

Thedifficultywithusingtheformulaisestablishingthevalueforr.Typically,therearethreervaluesgiven(bythemanufacturer)forarotor:themaximum,minimumandaverager.Thesecorrespondtothedistancesfromthecenterofrotationtothebottom,topandmiddleofthesampletube.

Ifthedensityandviscosityofthemediumareknown,aswellasthedensityofagivenparticle,thenthetimeneededtocompletelysediementaparticlecanbedeterminedbytheformula:

T=((D-L)/(D+L))*(N/(d(g-p)S))whereT=timeinminutesD=radialdistanceincmforrL=radialdistancetomeniscusN=viscosityofthefluidmediumg=densityofthefluidmediump=densityoftheparticletosedimentd=diameteroftheparticleincms.S=rotationalvelocityinRPM

ROTORCOMPARISONS

WorkingwiththemaximumRCFforeachrotor,theconversioncanbemadewiththefollowingequation:

t=(txRCF)/RCFwheret=runtimeneededfortheBeckmanrotort=runtimespecifiedintheprocedureRCF=RCFofJA-20rotoratmaximumspeedRCF=RCFspecifiedintheprocedure(calculatedifprocedureinRPM)

Thisequationcanbealteredsomewhattobeevenmoreuseful.Theefficiencyofrotorscanbecomparedforanygiventask.Eachrotorisgivenavaluefork(theclearingconstant,orclearingfactor),whichisanestimateofthetime(inhours)requiredtopelletaparticleofknownsedimentationcoefficientatthemaximumspeedoftherotor.4Thelowerthevalueofk,theshorterthetimerequiredtosedimentagivenparticle.Therelationshipisgivenby

t=k/s¸k=clearingfactorexpressedinhour-Svedbergss¸=SedimentationCoefficientinwaterat20°CexpressedinSvedbergs

Itispossibletocomputek,butitiseasiesttousethemanufacturersvalues,whichcanbelookedupintablessuchasthoseinTableF.1.

k=(253,300)[ln(r/r)]/(Rotorangularvelocity/1000)

Usingtabulatedkvalues,however,thecomparisonofrotorsiseveneasier,astheequationabovebecomes

t=kt/kwherethetandkvaluesarethetimeandkfactorforeachrotor.

Bysubstitutingthisequation,youcanalsodeterminethetimeofcentrifugationforanychangeinrotorspeed.5

ForaSorvallSS-34,forexample,at20,000RPM(k=402),thetimerequiredtosedimentaparticleof100S,inwaterat20°Ciscalculatedby:

t=402/100or4.02hours(4hrs,1min)

TableF.2presentsthekvaluesfortheSorvallSS-34rotorandfortheSorvallGSArotor.Sincethesetworotorswillbeusedthroughouttheremainderofthecourse,referenceshouldbemadetothistableinanyfuturecentrifugationwork,whenanotherrotorissubstituted.

ROTORSAFETY

Thereareanumberofsafetyprecautionsthatmustbeadheredtowhenusinganycentrifugeandrotor.

RotorFailure:

Allrotorsaresubjecttostressandwithtimewillundergometalfatigue.Thisisagiven,andconsequently,adetailedhistoryoftherotoruseshouldbekept.Thisisusuallynotdonewithclinicalcentrifuges,butisanabsoluteforanultracentrifugerotor.

EVERYUSEOFANULTRACENTRIFUGEROTORMUSTBERECORDEDINTHECENTRIFUGELOG.ABSOSLUTELYNOEXCEPTIONS!

Afteraperiodofuse,eachrotorwillinturnbederated,thatisitsmaximumRPMwillbelowered.TheBeckmanrotorsmaycontainopticalspeedcontrolringsattheirbase-besuretheyarepresent,andcleanbeforeuse.Thesedeviceswillstrobiscopicallymonitorthemaximumspeedthatarotorcanbeusedat.Theyarereplacedastherotorisderated.

Byfarthemostcommoncauseofrotorfailureiscorrosionstress.Salts,highlyalkalinedetergentsandofcoursecorrosiveacidsandalkali"swillcausedecompositionofthecoatingsonaluminumrotors,whichinturnwillconcentratestressandeventuallyresultincracksandtotalrotorfailure.Titaniumrotorsaremorecorrosionresistant,butmoreexpensive.Ultracentrifugerotorsareexpensive(inexcessof$6,000eachonaverage)andcanbepotentiallyhazardous.Attheforcesgeneratedinanultracentrifuge,arotorfailureistheequivalentofasmallbomb.

THEREFORETHEFOLLOWINGRULESMUSTBEOBSERVED.

1. Beforerunningacentrifuge,checktheclassificationdecalonthecentrifugetoensurethattherotorissafetouseinthecentrifugeathand.

2. Neveruseanalkalidetergentonarotor(mostarehighlyalkaline-besuretocheckbeforeuse).

3. Alwayscleanandcompletelydrytherotoraftereveryuse.Anyspilledmaterials,especiallysaltsandcorrosivesolventsmustberemovedimmediatelywithrunningwater.Fixedanglerotorsarestoredupsidedown,todrainafterthoroughcleaningandrinsing.Swingingbucketshaveonlythebucketscleanedanddried,andstoredinvertedandwiththecapsremoved.NEVERimmersetherotorportionofaswingingbucketrotor.Inevitablythelinkagepinswillrust,asitisvirtuallyimpossibletoremoveallfluidsfromthem.

4. Beespeciallycarefulnottoscratchthesurfaceofarotororbucket.Useplasticbrushesonly.Normalwirebrusheswillscratchtheanodizedsurfaceofaluminumrotorswhichwillincreasethelikelihoodofcorrosion.Theanodizedlayerisextremelythinandisthemaindefenseagainstcorrosionofanaluminumrotor.

5. Alwaysusethepropercentrifugetube.Glasstubesareusedinclinicalcentrifugesonly.HighSpeedCorextubescanbeusedupto15,000RPM(inSS34rotor)IFtherearenoscratchesorimperfectionsintheglass,andiftheproperrubberorplasticadapterisemployed.Allultracentrifugationuseemploysnitrocelluloseorpolyallomertubes.Nitrocelluloseagesandwillcollapseinastrongcentrifugalfieldifold.

6. Alwaysfillthecentrifugetubestotheproperlevel.(usuallyfulltowithin1/2inchofthetop).Thetubesarethinwalledandwillcollapseifimproperlyfilled.

7. Alwaysbalancetherotorproperly.Useaprecisionscaleformostwork.Alwaysbalancethetubewithamediumthatisidenticaltothatbeingcentrifuged,i.e.donotbalanceanalcoholsolutionwithwater,oradensesucrosesolutionwithwateronly--thedistributionofthedensitieswillbeincorrect.Forswingingbuckets,besurethebucketsareweighedwiththeircapsinplace,thatthesealsareintactandthatthecapsaresecure.Becarefulintheplacementoftubeswithinarotortoensureproperbalance-checkthemanufacturersguidesforcomplexrotorsthatholdmultipletubes.

8. Ensurethattherotorisproperlyseatedwithinthecentrifuge.Forswingingbuckets,ensurethattheyarehangingproperly-DoubleorTriplecheck!Forpreparativerotors,besuretherotorcoverisinplaceandproperlyscreweddown,whereappropriate.NEVERusearotorwithoutitslid,whenoneissupplied-thescrewactuallyholdstherotortothemotorshaft.

9. Checkthatthecentrifugechamberisclean,defrostedandthatallmembranesormeasuringdevicesareintactandfunctional(Beckmanspeedandtemperaturecontrols)andthatthelidissecurelyclosed.

10. Adjustaccelerationrates,decelerationrates,temperatureandRPMcontrolsasappropriate.Setbrakeonoroffasappropriateandcheckvacuumlevelwhereappropriate.

11. Startthecentrifugeandsetthetimer.Donotattempttoopenthecentrifugeuntiltherotorhascometoacompletestop.

12. Beforeopeningthecentrifuge,recordtheappropriateinformationinthecentrifugelog.

Note:Ifproperlybalancedandused,therotorshouldacceleratesmoothlyandwithaconstantchangeinthepitchofthemotorsound.Anyvibrations,orunusualsoundsshouldcausethecessationofoperationIMMEDIATELYbytheoperator.NEVERleavethecentrifugeuntilyouarecertainthatithasreacheditsoperatingspeedandisfunctioningproperly.Allrotorsgothroughaminorvibrationphasewhentheyfirststart.Therewillbeaminorflutterwhentherotorreachesthisvibrationpoint-donotconfusethiswithaseriousvibrationcausedbyimbalance.Ifindoubt,haltthecentrifugeandgetassistance.