CentrifugationisaprocessusedtoseparateorconcentratematerialssUSPendedinaliquidmedium.Thetheoreticalbasisofthistechniqueistheeffectofgravityonparticles(includingmacromolecules)insuspension.Twoparticlesofdifferentmasseswillsettleinatubeatdifferentratesinresponsetogravity.Centrifugalforce(measuredasxg,gravity)isusedtoincreasethissettlingrateinaninstrumentcalledacentrifuge.Twocommonexamplesoftheuseofcentrifugalforceare:(1)Whenyoudothe"aroundtheworld"trickwithayo-yo,itiscentrifugalforcethatmakestheyo-yobodystayattheendofthestringasyourotateit;and(2)Whenyouwashclothesinawashingmachine,itiscentrifugalforcegeneratedinthe"spin"cyclethatforceswateroutofthefabrictofacilitatefasterdrying.

Centrifugesaredevicesusedinavarietyofscientificandtechnicalapplicationswhichspincarriervessels(centrifugetubes)athighrotationspeedsandveryhighcentrifugalforce.Thecentrifugalforce(expressedas#gravitiesor,#xg)generatedisproportionaltotherotationrateoftherotor(inrpm)andthedistancebetweentherotorcenterandthecentrifugetube.Therefore,agivencentrifugemayusemultiplerotorsizestogiveflexibiltyinchoosingcentrifugationconditions.Eachcentrifugehasaspecialgraph,anomograph,oratablewhichrelatesrotationrate(rpm)tocentrifugalforce(xg)foreachsizeofrotoritaccepts.

Typically,thematerialtobe"spun"isplacedinacentrifugetubewhichisthenplacedinarotor.Therotorisagenerallyadensemetalwhichdissipatesheatquickly,andisofsufficientmassthatitgeneratesmomentum,i.e.,onceitsspinningitrequireslittleenergytokeepitgoing.Centrifugesgenerallyworkundervacuumandarerefrigeratedtoreduceheatingcausedbyfrictionalforcesastherotorspins.Rotorsareusuallystoredinrefrigerationunitstokeepthematorneartheoperatingtemperature.

Becausecentrifugescomeinallshapesandsizes,andtherotorsvary,theuniversalandtransferableunitofcentrifugationiscentrifugalforce(xg).Inlabwrite-upsyoushouldreportthecentrifugationforceused(#gravities)becauseitisthetransferableunitbetweendifferentcentrifuges.

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DifferentialCentrifugation

Themostcommontechnique,calleddifferentialcentrifugation,isusedtoseparateparticlesfromaliquidmediumortoseparateparticlesofdifferentmassesintoseparatefractionsofthesupernatant.Wewillusethistechniqueinaseveralwaysinthiscourse.

1.IntheBios42Amylaselabwewillusecentrifugationtopelletthecellulardebrisandexcessstarchduringtheenzymeextractpreparation.Theenzyme,whichissoluble,willremaininthesupernatant.Duringtheactualexperiment,wewillusecentrifugationtoseparatetheenzyme(soluble)fromitssubstrate(insolubleamylose-azure)tostopthereaction.

2.Inthemolecularlabswewillusecentrifugationtopromoteachemicalreactionbyforcingsmallquantitiesofreactantstogetherinthebottomofmicrocentrifugetubes.Wewillalsousecentrifugationtopreparebacterialcellsfortransformationbyalternatelypelletingthemandthenresuspendingthemwithdifferentchemicalsolutions.

3.IntheHillReactionlabwewilluseamulti-stepdifferentialcentrifugation(Fig.9-3)toisolatecellorganelles(chloroplasts)fromcrudecellularhomogenate.Becausetheorganelleshavemuchlessmassthanthecellwallcomponents,thefirstpelletthatformsatlowcentrifugalforceisprimarilycellulardebris.Theorganellefractionisthenpelletedathighercentrifugalforce.

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CentrifugeCautions:

ThesecautionspresumeyouhavehadproperinstructionintheuseofthecentrifugeORhavereadtheinstructionmanualthoroughly.

1.Makesurethecorrectrotorisbeingusedandthatitisinstalledproperlyonthespindle.

2.Balancetheloadintherotor-everytubemusthaveabalancetubeintheoppositeslotwiththesamevolumeoffluid.

3.Makesureyouareusingtheappropriatecentrifugetubeforthejob-theycanruptureattoohighaspeed.

4.Pre-coolthecentrifugeandtherotor.Rotorsshouldbestoredinarefrigerator.

5.DONOTattempttooverrideanysafetyfeaturesofthecentrifuge.

6.NEVERleavethecentrifugeunattendeduntilitreachesmaximumspeedandisgoingsmoothly.

7.Whenindoubt,ASKFORHELP.