离心机参数计算

CentrifugationTheoryandPractice•Routinecentrifugerotors•Calculationofg-force•Differentialcentrifugation•DensitygradienttheoryCentrifugerotorsFixed-angleaxisofrotationAtrestSwinging-bucketgSpinninggGeometryofrotorsbcrmaxravrminrmaxravrminSedimentationpathlengthaxisofrotationarmaxravrmink’-factorofrotors•Thek’-factorisameasureofthetimetakenforaparticletosedimentthroughasucrosegradient•ThemostefficientrotorswhichoperateatahighRCFandhavealowsedimentationpathlengththereforehavethelowestk’-factors•Thecentrifugationtimes(t)andk’-factorsfortwodifferentrotors(1and2)arerelatedby:2211ktktCalculationofRCFandQ2100018.11QrxRCFrRCFQ299RCF=RelativeCentrifugalForce(g-force)Q=rpm;r=radiusincmRCFinswinging-bucketandfixed-anglerotorsat40,000rpm•BeckmanSW41swinging-bucket(13ml)•gmin=119,850g;gav=196,770g;•gmax=273,690g•Beckman70.1Tifixed-anglerotor(13ml)•gmin=72,450g;gav=109,120g;•gmax=146,680ggdvlp18)(2Velocityofsedimentationofaparticlev=velocityofsedimentationd=diameterofparticlep=densityofparticlel=densityofliquid=viscosityofliquidg=centrifugalforceDifferentialcentrifugation•Densityofliquidisuniform•DensityofliquidDensityofparticles•Viscosityoftheliquidislow•Consequence:Rateofparticlesedimentationdependsmainlyonitssizeandtheappliedg-force.Sizeofmajorcellorganelles•Nucleus4-12m•Plasmamembranesheets3-20m•Golgitubules1-2m•Mitochondria0.4-2.5m•Lysosomes/peroxisomes0.4-0.8m•Microsomalvesicles0.05-0.3mDifferentialcentrifugationofatissuehomogenate(I)1000g/10minDecantsupernatant3000g/10minetc.Differentialcentrifugationofatissuehomogenate(II)1.Homogenate–1000gfor10min2.Supernatantfrom1–3000gfor10min3.Supernatantfrom2–15,000gfor15min4.Supernatantfrom3–100,000gfor45min•Pellet1–nuclear•Pellet2–“heavy”mitochondrial•Pellet3–“light”mitochondrial•Pellet4–microsomalDifferentialcentrifugation(III)Expectedcontentofpellets•1000gpellet:nuclei,plasmamembranesheets•3000gpellet:largemitochondria,Golgitubules•15,000gpellet:smallmitochondria,lysosomes,peroxisomes•100,000gpellet:microsomesDifferentialcentrifugation(IV)•Poorresolutionandrecoverybecauseof:•Particlesizeheterogeneity•ParticlesstartingoutatrminhavefurthesttotravelbutinitiallyexperiencelowestRCF•SmallerparticlesclosetormaxhaveonlyashortdistancetotravelandexperiencethehighestRCFDifferentialcentrifugation(V)Fixed-anglerotor:ShortersedimentationpathlengthgmaxgminSwinging-bucketrotor:LongsedimentationpathlengthgmaxgminDifferentialcentrifugation(VI)•Rateofsedimentationcanbemodulatedbyparticledensity•NucleihaveanunusuallyrapidsedimentationratebecauseoftheirsizeANDhighdensity•Golgitubulesdonotsedimentat3000g,inspiteoftheirsize:theyhaveanunusuallylowsedimentationratebecauseoftheirverylowdensity:(p-l)becomesratelimiting.DensityBarrierDiscontinuousContinuousDensitygradientcentrifugationHowdoesagradientseparatedifferentparticles?LeastdenseMostdensegdvlp18)(2Whenpl:vis+veWhenp=l:vis0Predictionsfromequation(I)gdvlp18)(2Whenpl:vis-vePredictionsfromequation(II)Summaryofpreviousslides•Aparticlewillsedimentthroughasolutionifparticledensitysolutiondensity•Ifparticledensitysolutiondensity,particlewillfloatthroughsolution•Whenparticledensity=solutiondensitytheparticlestopsedimentingorfloatingBuoyantdensitybandingEquilibriumdensitybandingIsopycnicbanding152341233FormatsforseparationofparticlesaccordingtotheirdensityWhendensityofparticledensityofliquidVis-veDiscontinuousResolutionofdensitygradientsContinuousDensityBarrierIIIProblemswithtoploadingpl:vis+veforallparticlesthroughoutthegradientSeparationofparticlesaccordingtosize