生物分离工程 课件-双语 chapter 4 precipitation and crystallization

生物反应器细胞收集细胞破碎沉淀结晶纯化产品制剂BioreactorExtractionCelldisruptionCellharvestingPurificationProductformulationPrecipitationandcrystallizationSupernatantPrecipitationandcrystallizationSolutionColloidChapter4

PrecipitationandCrystallization4.1Precipitation(沉淀)4.2Crystallization(结晶)沉淀：得到无定形物质(Amorphous)的过程。结晶：形成晶形物质(crystal)的过程。4.1precipitationIntroductionSaltingoutOrganicsolventprecipitationIsoelectricprecipitationPrecipitationbytheadditionofnon-ionicpolymersSelectivedenaturationIntroductionPrecipitation

theconversionofthesolublesolutesintoinsolublesolids.oftenusedinthepreliminarystagesofdownstreamprocessing.Oftenusedfortherecoverybulkproteins.Precipitatesgenerallydonothavearegularmorphology.ProteinprecipitationmethodsAdvantagesProteinprecipitationmethodsCanbeclassifiedbroadlyintotwogroups:(1)precipitationbasedonsolventpropertymodificationpHchange(isoelectricprecipitation等电点沉淀)Ionicstrengthchange(saltingout盐析)Changeindielectricconstant介电常数

(organicsolventmediatedprecipitation)Reducetheamountof

wateravailabletointeractwiththeprotien

(precipitationbynon-ionicpolymers)Proteinprecipitationmethods(2)precipitationbasedonsolutepropertymodificationSelectiveinteraction

withmetalpolyelectrolytes(聚合高分子电解质)oraffinityreagentsSelectivedenaturation

toprecipitateunwantedproteinspHdenaturationThermaldenaturationOrganicsolventdenaturationAdvantagesReductioninthevolumeoffermentationbroth.ConcentrationofthedesiredproductRapidseparationandstabilizationparticularlyoflabileproductsThemethodologyandequipmentaresimpleSaltingoutSaltinginBasicprincipleofsaltingoutFactorsinfluencingsaltingouteffectProteinconcentration,salttypeandsaltconcentration,pHvalue,temperature

Ammoniumsulfateprecipitation0.001M0.005M0.01M0.02M[NaCl]pIpH>5.2pH=5.24.85.05.25.45.6pH0.0010.010.1NaClconcentration(M)solubilitySaltingin盐溶BasicprincipleofsaltingoutRemovalof'bound'watermolecules.ChargeneutralizationCoagulate(凝结）byforminghydrophobic(疏水作用)interactionswitheachother.

中性盐中和其电荷中性盐中和其电荷SO42-等NH4+或Na+等－－－＋－＋＋＋－－－－－－－－OH-H+＋＋＋＋＋＋＋＋H+OH-pH<pI，带正电荷，又有水膜，是稳定的亲水胶体pH=pI，处于等电点状态，水膜未脱，是不稳定的亲水胶体pH>pI，带负电荷，又有水膜，是稳定的亲水胶体中性盐破坏水膜中性盐破坏水膜中性盐破坏水膜＋＋＋＋＋＋＋＋－－－－－－－－AmmoniumsulfateprecipitationAdvantagesDisadvantagesApplicationsProceduresIncreasingsaltconcentrationsDecreasingsaltconcentrationsPilotexperimentammoniumsulfateprecipitationPreparativeexperimentammoniumsulfateprecipitationAdvantageslargesolubilityinwatersalting-outeffectislessdependentontemperatureintherange0-30℃.2-3Mammoniumsulfatesuspensionofproteinprecipitateorcrystalsisoftenstable

foryearsthehighsaltconcentrationpreventsproteolysisandbacterialaction.becomethenormalpackagingmethodforcommercialenzymes.AdvantagesAmmoniumsulfatecan

beremovedbydialysis,ultrafiltrationorgelfiltration.

Lowheatofsolubilization(溶解）preventsdenaturation变性ofproteinsLowdensityofsaturatedsolutions(1.25g/cm3)proteinscanbecollectedinpelletsbycentrifugationCheapDisadvantagesUndergoeshydrolysisreleasingammoniaathigherpHvaluesBeingcorrosive(腐蚀性)Residuesofammoniumsulfateremaininginfoodproductscanaffectthetasteevenatlowconcentrationsitisalsotoxicwithrespecttoclinicaluseandhencemustberemoved.ApplicationsConcentrationofproteinsbybulkprecipitationPurificationofproteinsbyfractionationduetodifferencesinsolubilityIncreasingsaltconcentrationseitherasaturated(=100%,w/v)saltsolutionorpowderedsaltcrystalsareslowlyaddedtotheproteinmixturetobringupthesaltconcentrationofthemixture.Note:saturationlevelisdependenton

temperature!!在进行硫酸铵盐析时，一般用饱和度表示其浓度，将达到饱和状态的硫酸铵的饱和度定为100%.25oC时，硫酸铵的饱和溶解度是767g/L，定义为100%饱和度DecreasingsaltconcentrationsFirst，precipitateproteinsasmuchaspossiblewithaconcentratedsaltsolution.Thenaseriesofcold(near0℃)ammoniumsulfatesolutionsofdecreasingconcentrationsareemployedtoextractselectivelytheproteincomponents.Pilotexperimentammoniumsulfateprecipitation1.1mlproteinsamplesonice(appr.10samples)2.Addsolidammoniumsulfateto0,10%,20%,30%....90%saturation.Mixwell,untila.s.isfullydissolved.3.Leaveonice(>30min).4.Centrifuge,10min，13000rpm,coldroom5.Lookforprecipitates.Transfersupernatantstocleantubesandmeasure

activityThisproteinprecipitatesbetween40%and70%ammoniumsulfatesaturation(‘fractionationrange’).x=40%;y=70%inthisexample.Preparativeammoniumsulfatefractionation1.Measurevolumeofproteinsolution(M1).Addammoniumsulfate

to40%saturation饱和度,equilibrate平衡,centrifuge.2.Collectsupernatant上清液(S1)andkeepthepellet沉淀(P1).Measurevolumeofsupernatantagain,addammoniumsulfateto70%saturation,equilibrate,centrifuge.3.Collectpellet(P2)anddissolve溶解P2inbuffer缓冲液M1S1P1P2S2OrganicsolventprecipitationBasicprinciple(seeP42-43)DisadvantagesSelectioncriteriaFactorsinfluencingtheeffectoforganicsolventprecipitationMacromoleculeconcentration,organicsolventtypesandconcentration,pH,temperature,ionicstrength,metalionsProcedure

DisadvantagesDenaturingeffectoforganicsolventsonproteins,particularlyathigherconcentrationsandattemperaturesgreaterthan10℃.Organicsolventsareinflammable(易燃的)necessitatingtheuseofcostlyflameproofequipment

（防爆设备）inlargescaleoperations.SelectioncriteriaBeingwater-miscible(混溶的)organicsolventsDonotreactwithproteinsHavinggoodprecipitatingeffectBeingnon-toxicandlowflammabilityEthanolandacetonearethemostwidelyusedsolvents.Procedure①在4℃以下，按一定比例向蛋白溶液中缓慢加入冰冷（-20℃）的有机溶剂（如丙酮或乙醇），在冰浴中持续温和地搅拌；②溶剂加完后，继续在冰浴中搅拌10－15min；③低温离心：10,000g，10min；④除去上清；⑤用2倍沉淀体积的冷的缓冲液溶解沉淀阴离子交换层析阳离子交换层析凝胶过滤脱盐脱脂超滤NaturalMineralCrystalNaturalMineralCrystalDiamond&graphiteCrystalInDailyLifeSaltSugarMSG

monosodiumglutamateICECrystalstructureUnitcell晶胞晶格点Acrystalisasolidinwhichtheconstituentatoms,molecules,orionsarepackedinaregularlyordered,repeatingpatternextendinginallthreespatialdimensions.InternalstructureofcrystalsaccessiblebyX-raydiffraction(X射线衍射)analysis.WhyuseX-raydiffraction？光波与可测量物体的大小X-raydiffractionMechanismandProcedureofX-raydiffractionDNAdoublehelixandx-raydiffractionEquipment4.2Crystallization4.2.1crystallizationprocessCharacteristics(seeP49)CrystallizationtheoryCrystallizationprocessImprovementofthecrystalquality4.2.2CrystallizerBatchoperationContinuousoperation4.2.3ProteincrystallizationInsulincrystalsCrystallizationtheorySaturation

Saturationisthemaximumconcentrationofthesolutethatisthermodynamicallystableinsolution.

Supersaturation

thesolutioncontainmoresolutethanthatispresentatsaturation.Supersaturatedsolutionsarethermodynamicallyunstable.solubility-temperaturephasediagramnewnucleiformspontaneously自发产生新晶核Growthofexistingcrystal,formationofnewnucleiNocrystallizationoccurs无结晶solubility-temperaturephasediagramsupersolubilitycurve超溶解度曲线solubilitycurve溶解度曲线亚稳区稳定区不稳区第一亚稳区：加入晶种结晶会生长，但不产新晶核；第二亚稳区：加入晶种结晶会生长，同时有新晶核产生第一亚稳区第二亚稳区metastablezone(亚稳区）Asolutionmaymaintainitssupersaturationoveraconcentrationrangeforacertainperiodwithouttheformationofasecondaryphase.Thisregioniscalledthemetastablezone.inductiontimeFromthecreationofsupersaturationtothefirstappearanceofthesecondary(solid)phase,thetimeelapsediscalledinductiontime.Assupersaturationincreases,theinductiontimeisreduced.themetastablezonewidthWhenthesupersaturationreachesacertainlevel,theformationofthesecondaryphasebecomesspontaneousassoonassupersaturationisgenerated.Thispointisdefinedasthemetastablezonewidth.Crystallizationprocessnucleation

Intheabsenceofanysolidparticle,nucleationmustoccurbeforecrystalgrowth.drivingforce：supersaturationcrystalgrowthdrivingforce：supersaturationGenerationofSupersaturationCooling(withsomeexceptions)EvaporationVacuumcoolingChemicalReactionSaltingoutprimarynucleation初次成核:nucleationincrystalfreesystemsecondarynucleation二次成核:inducedbythepresenceofcrystals

homogeneousnucleation均相成核:spontaneous自发的Heterogeneousnucleation异相成核:inducedbythepresenceofforeignparticlesNucleation成核Inindustrialprocesses,homogeneousnucleation均相成核israreNucleationisusuallyheterogeneous异相的

and/orsecondary二次的NucleationHomogenousnucleation均相成核asaresultofsupersaturation过饱和only,andrequiresaperfectlycleanvessel容器withnoroughsurfacesHeterogeneousnucleation异相成核causedbydust,dirt,roughspotsonwalls,etcsecondarynuclei

Contactnucleationisthemostimportantsource.causedbyinteractionofexistingcrystalswithvessel,impellerorbycollisions.CrystalgrowthNucleationbirthofanewcrystalCrystalgrowthnucleitogrowlargerbytheadditionofsolutemoleculesfromsupersaturatedsolution.Crystalgrowth,alongwithnucleation,controlsthefinalparticlesizedistribution.

Theconditionsandrateofcrystalgrowthhaveasignificantimpactontheproductpurityandthecrystalhabit（晶体惯态）.MoleculesinsolutionMoleculesincrystalTransportProcessesDuringCrystallizationImprovementofthecrystalqualityCrystalqualityCrystalsizesupersaturation,coolingvelocity,stirvelocity,seedcrystalCrystalformandmorphologySupersaturation,solvent,impuritiesCrystalpurityImpurities,crystalsizedistribution,crystalhabitCrystalcakingRecrystallizationL-glutamicacid谷氨酸α-form结晶呈颗粒状，产品纯度高；β-form结晶呈片状、针状，比表面积大，易包含母液和杂质。分批（间歇）结晶Operationprocess结晶器的清洁加入固料或料液到结晶器用任何合适的方法产生过饱和度成核和晶体生长晶体的排除操作要点：为了控制晶体的生长，获得粒度较均匀的产品，必须尽一切可能防止不需要的晶核生长。缓慢冷却，将溶液状态控制在介稳区内；在适当时机加入适量晶种；温和搅拌，尽量避免二次成核现象。分批（间歇）结晶优点：结晶设备简单；结晶条件容易控制；获得的结晶体纯度高，而且粒度分布均匀；尤其适合产品浓度较低、粘度大、杂质多的系统——如：发酵产品的结晶。缺点：操作成本比较高，操作和产品质量的稳定性较差。分批（间歇）结晶结晶设备：立式搅拌结晶罐：操作容易，搅拌较慢（30rpm）（如谷氨酸和柠檬酸结晶）。卧式结晶槽：容积较大，搅拌速度很慢（<10rpm），晶体不易破碎（可作为味精结晶时的助晶槽）。真空结晶器：结晶速度快，容易自然起晶，适合产品要求颗粒粗大时使用（如带一个结晶水的谷氨酸钠盐）。连续结晶优点：(1)冷却法及蒸发法(真空冷却法除外)采用连续结晶操作费用低，经济性好。(2)结晶工艺简化，相对容易保证质量。(3)生产周期短，节约劳动力费用。(4)结晶设备的生产能力可比分批操作提高数倍甚至数十倍。(5)易于实现自动化控制连续结晶操作缺陷:成核速率难以控制晶核生长速率过高:晶体平均粒度过小，粒度分布过宽；结晶率下降。克服操作缺陷的方法细晶消除根据“淘析”原理，在结晶器中设立一个“澄清区”。将大小晶粒分开，大颗粒回到结晶器的主体部分，继续长大；小颗粒进入细晶消除系统，加热或稀释使之溶解后，重新回到结晶器中。产品粒度分布排料产品经过一个“分级排料器”，不符合要求的晶体被截流后返回结晶器继续长大。清母液溢流根据密度不同将含有细晶的母液通过溢流而排出，从而提高结晶器内晶浆的密度。连续结晶结晶设备：Oslo型结晶器：过饱和产生区和晶体生长区是分开的，晶体在循环母液流中流化悬浮，为晶体生长提供了有利条件，产生的晶体大而均匀。DTB型结晶器：有澄清区，可进行细晶消除；有淘析腿，可进行产品粒度分布排料；结晶器内设有导流筒，可进行清母液溢流操作。设有内、外循环通道，有利于晶体的均匀生长，且底部不易结疤。Example1SterileCrystallizationofImipenem（亚硫胺霉素）Goal:AchievedesiredphysicalpropertiesandchemicalpurityIssues:Sterileoperation,narrowparticlesizedistribution(PSD),smallmeanparticlesize(20microns)satisfytherapiddissolutionrequirement:<20secondsinanaqueoussolutionPreparationofanaqueoussolutionIssue:

Lowsolubilityinwateratoperatingtemperatures.ThesolubilitycurveisshowninFig.11-1.FeedPreparation,Option1Concentration:10g/literBeconcentratedto30g/literbyreverseosmosissterilefiltrationadditionofacetonecrystallizeFigure11-2FlowsheetforExample1;feedpreparationandcrystallizationofanantibioticundersterileconditions.FeedPreparation,Option260g/literat60℃contacttime:short,about1minutetotalCrystallizationTheimipenemcannowbecrystallizedbytheadditionofacetone.Option1:SlowAdditionwithoutSeedOption2:AdditionofAcetonewithSeedingandControlledGrowthOption3:UseofMilledSeedFinalProcessAcetone,7%byvolume,isaddedtothesterile-filteredaqueoussolution.Milledseed(0.4%)isadded.Theremainingacetoneisadde