生物分离工程 课件汇 -双语 Chapter 6-Extraction-1-Organic solvent extraction -chapter 9 electrophoresis

Chapter

7

Extraction1IntroductionMosquitoAfrica

DiseaseHerbWhatcomestoyourmind?2IntroductionTuYouyouextracted

artemisininfromArtemisiaannua

totreatmalaria.How

to

extract

artemisinin？3ThinkingWhatisthebasicprincipleofextraction？HowtoproposearationalextractionprocessHowtoimprovetheextractionefficiencyofartemisinin？4Solventextraction

isamethodofseparationbasedonthetransferofasolutefromonesolventintoanothersolventwhenthetwosolventsarebroughtintocontact.Itisessentialthatthetwosolventsbeimmiscible不混溶的.Asolutewhichissolubleinbothphases相willdistributebetweenthetwophases两相inadefiniteproportion特定的比例.Principle5溶质2溶质1原溶剂（重相）萃取剂（轻相）6PrincipleOrganicsolventextractionReversemicellesextractionAqueoustwo-phaseextractionSupercriticalfluidextraction7ClassificationofextractiontechniquesOrganicsolventextraction(有机溶剂萃取)Reversemicellesextraction(反胶束萃取)Aqueoustwo-phaseextraction(双水相萃取)Supercriticalfluidextraction(超临界流体萃取)8ClassificationofextractiontechniquesOrganic

Solvent

ExtractionPrincipleCharacteristicsOperationprocessFactorsinfluencingseparationeffectExtractionequipmentsApplication9DistributioncoefficientDistributionratioExtractionfactor

Extraction

efficiency

SeparationfactorDefinition10Distributioncoefficient分配系数Distributionratio分配比Extractionfactor萃取因素/萃取比Extraction

efficiency萃取率Separationfactor分离因数Definition11DistributioncoefficientThepartitioningofthesolutebetweenthetwophasesisexpressedquantitativelybypartitioncoefficientordistributioncoefficient.Thepartitioncoefficient,K,istheratioofthesoluteconcentrationintheextractphase(x)tothatintheraffinatephase

(y)atequilibriumconditions.K=x/y12K=x/y=?21VxVYK==28/22/1XY=Distributioncoefficient13ThevalueofKisindependentofthesoluteconcentrationforagivensolventpairandisaconstantatagiventemperature.Requirement：Diluted

solutionThesolutehasnoeffectonsolventsolubility.Itmustbethesamemoleculartypeandnocomplexationordissociationoccurs.Distributioncoefficient14DistributionratioThedistributionratio,D,istheratioofthetotalconcentrationofspeciesofasoluteintheextractphasetothatintheraffinatephase.D=CL/CR=(CL1+CL2+CL3+···+CLn)/(CR1+CR2+CR3+···+CRn)CLCRCL1CR1CL2CR2CL3CR315DistributionratioD==21VLVR1LR1111111112222233333=CLCRCL1+CL2+··+CLnCR1+CR2+··CRnD==24+2+22+1+116DistributionratioAssumingthereisnoassociation,dissociationorpolymerizationinthebothphasesthen,underidealizedconditions,KwouldbeequaltoD.17ExtractionfactorExtractionfactoristheratioofthetotalmassofasoluteintheextractphasetothatintheraffinatephase.E=M1V1/M2V2=D×V1/V221V1V21121111111112222233333=D==24+2+22+1+1E==D×M1V1M2V2V1V2E=D×2=418Inactualproduction,theextractionefficiencyisoftenusedtorepresenttheextractionabilityofanextractantforacertainsolute.Itisdefinedasthepercentageoftheamountofsoluteintheextractphasetotheamountofsoluteinthefeedsolution.：Extraction

efficiencyΗ=萃取相中溶质总量/原料液中溶质总量×100%=M1V1/(M1V1+M2V2)×100%=E/(E+1)×100%19Extraction

efficiency1121111111112222233333E=D×2=4H=E/(E+1)×100%=4/(4+1)

×100%=80%20SeparationfactorTheselectivityofextractionbetweentwosolutecomponentsisgivenbyseparationfactor,β,astheratioofthedistributioncoefficientsofthetwosolutesAandB.β=(

c1A/c1B)/（

c2A/c2B)=

(

c1A/

c2A)/（

c1B/c2B)=KA/KB21SeparationfactorAAAAAAAAAA21V1V2=12BBBBβ=KA/KBβ==421/222SeparationfactorIfAisthetargetproductandBistheimpurity,theseparationfactoris:β=Kprod=Kimp=

K产/K杂Thelargertheβ,theeasieritistoseparate；Kprod=Kimp,β=1,twosubstancescannotbeseparated.Theseparationfactorvarieswiththeconcentration,composition,aqueousphasecompositionandtemperature.Theseparationfactordeterminesthepurity,andtheextractionpercentage

determinesthe

yield.23Extraction:SequenceofeventsMixingorcontactingintimatelymixingthetwosolventsuntilthesolutehasbeendistributed分配betweentheliquidsPhaseseparation相分离Collectionofseparatephases分离相的收集24Removebiochemicalsfromtheorganicphase(1)Evaporation蒸发，Crystallization结晶

fromtheorganicphase(2)Back-extraction反萃取

intoaqueousphase水相followedbyfurtherpurification纯化andthencrystallization结晶.Extraction:Sequenceofevents25DiscussionDraw

an

extractionprocessforextractingartemisininfromArtemisiaannuajuice26？Initialconcentrations:

Artemisininintheaqueousphase:200mg/L

Chlorophyllintheaqueousphase:400mg/LAfterextraction:

Artemisininintheaqueousphase:160mg/L

Chlorophyllintheaqueousphase:340mg/LAssumenochangeinthevolumeofthetwophasesduringtheextractionprocess.Pleasecalculatethedistributioncoefficients,

extractionfactor,extractionefficiency,andseparationfactorsforartemisinin.Pleasedesignapotentialextractionprocess27青蒿收集、前处理氯仿混合萃取离心分离两相蒸发、干燥收集青蒿植物，粉碎，加水浸提用2倍体积的氯仿60℃萃取6小时，充分搅拌离心、过滤进行相分离，收集氯仿萃取相蒸发干燥去除氯仿，制得青蒿素粉末DiscussionDiscussion28But,

LowYield,LowActivity,andLowPurityBy

Kimi.

ExploreFactorsinfluencingseparationeffectpHTemperatureTimeSaltingoutSolventEmulsion29ThepHaffectsthedistributioncoefficientofweakacidsorweakbasesindrugs.ThepHaffectsthestability

of

the

compound

1.

pHFactorsinfluencingseparationeffect30

青霉素采用醋酸丁酯萃取时

pH=4.4不能进行

pH>4.4青霉素由有机相转入水相（反萃取）

pH<4.4青霉素被萃取到有机相1.

pHFactorsinfluencingseparationeffect31PenicillinpKa=4.4萃取相萃余相Time

(h)Reduced

Activity

(%)21.9642.5282.78245.32Temperaturehasagreatimpactontheextractionofbioactivesubstances,andgenerallyshouldbeextractedatlowtemperature.Theextractiontimealsoaffectsthestabilityofbioactivesubstances.Thepenicillinactivityinbutylacetatechangesovertime(atroomtemperature).Factorsinfluencingseparationeffect2.

Temperature

and

time32Saltingagents(suchassodiumchloride,ammoniumsulfate,etc.)bindtowatermoleculesandreducethesolubilityofsolutesinwater,makingthemmoresusceptibletomovetotheorganicphase.Water

solubility↓，K↑Saltingagentsincreasetherelativedensityoftheextractionphase,whichhelpsphaseseparation.However,itisimportanttousetheproperamountofsaltingagents,asexcessiveamountscancauseimpuritiestobetransferredtotheorganicphase.例：青霉素从水相→丁酯中，加氯化钠，提高质量和收率；分相容易。Factorsinfluencingseparationeffect3.

Saltingout33Factorsinfluencingseparationeffect4.

Solvent

1.

Highdistributioncoefficient

(相似相溶)2.

Separationfactorgreaterthan1(Extract

product)3.

Lowmutualsolubilitybetweenfeedandextractant4.

Lowtoxicity,

highstability,lowcorrosiveness,lowboilingpoint,lowvolatility,lowcost,convenientsource,andeasyrecyclability…34Factorsinfluencingseparationeffect5.

EmulsionEmulsionisaphenomenonofaliquiddispersed(dispersedphase)inanotherimmiscibleliquid(continuousphase).ItisdividedintoO/WandW/O.What’s

the

influence

?35Factorsinfluencingseparationeffect5.

EmulsionEmulsionisaphenomenonofaliquiddispersed(dispersedphase)inanotherimmiscibleliquid(continuousphase).ItisdividedintoO/WandW/O.Emulsion

makesitdifficulttoseparateorganicsolventphaseandaqueousphase:Aqueousphaseentrainmentoforganicphasemicrodroplets-affectingtheyield;Organicphaseentrainmentofaqueousphasemicrodroplets-affectingpurity

and

difficulttoseparateafterwards

.How

to

prevent

and

break

emulsion？36Factorsinfluencingseparationeffect5.

Emulsion—

Demulsification

(破乳化)Prevent:

Pre-treatmentandfiltration-toreducetheprotein

impurities;Break:

Addsurfactanttochangesurfacetensiontobreakupemulsion;Centrifugation;

Chemicalmethod:addelectrolyte(sodiumchloride,ammoniumsulfate,etc.)to

neutralizethechargeofthedispersedphaseintheemulsiontopromoteitsprecipitation;Physicalmethod:heating,dilution,etc.37Low

Yield,

Low

Activity,

Low

Purity38青蒿收集、前处理氯仿混合萃取离心分离两相蒸发、干燥收集青蒿植物，粉碎，加水浸提用2倍体积的氯仿60℃萃取6小时，充分搅拌离心、过滤进行相分离，收集氯仿萃取相蒸发干燥去除氯仿，制得青蒿素粉末DiscussionHow

did

Tu

Youyou

do

to

improve

extraction

efficiency？pHTemperatureTimeSaltingoutSolventEmulsionDiscussionMW=282.34pKa=4.5

39东晋葛洪《肘后备急方》:“青蒿一握，以水二升渍，绞取汁，尽服之”Pleasedesignapotentialextractionprocess40青蒿收集、前处理石油醚浸取乙醇萃取分相，收集蒸发、干燥收集青蒿植物，粉碎用60倍体积的石油醚30℃浸取24小时，过滤加入等体积乙醇萃取，去除叶绿素、蜡质等蒸发干燥去除石油醚，制得青蒿素粉末Discussion分液漏斗分相，收集石油醚相如何进一步提高萃取效率？Operation

Method（操作方式）Multi-Stage（多级萃取）Cross-current（多级错流萃取）Counter-current（多级逆流萃取）Single

Stage（单级萃取）Extraction

operation41Extraction

operation1.

Single-stageextraction:Onlyincludesonemixerandoneseparator.1.单级萃取42Extractionyield

(萃取率)

φResidualyield

(萃余率)1-φExtractionfactor

萃取因素Residualyield萃余率Extractionyield

萃取率Singlestageoperation43例：洁霉素在20℃

和pH10.0时分配系数（丁醇/水）为18。用等量的丁醇萃取料液中的洁霉素和用1/3体积的丁醇萃取其萃取率分别是多少？(1)E=K×1/1＝181-Ψ=18/(18+1)=94.7%(2)E=K×1/3/1=61-Ψ=6/(6+1)=85.7%当分配系数相同而萃取剂用量减少时，其萃取率下降。Singlestageoperation44Cross-currentoperation2.多级萃取—错流萃取45从哪里收集产物？经一级萃取后，萃余率φ1：经二级萃取后，萃余率φ2：经n级萃取后，萃余率：Extractionyield萃取率：Cross-currentoperation46例：红霉素在20℃

和pH9.8时分配系数（醋酸丁酯/水）为44.5。用1/2体积的醋酸丁酯进行单级萃取和二级错流萃取其萃取率分别是多少？(1)E=44.5×1/2/1=22.25

1-Ψ=22.25/(22.25+1)=95.7%(2)E=44.5×1/4/1=11.1251-Ψ=[(11.125+1)2-1]/(11.125+1)2=99.32%Cross-currentoperation47Cross-currentoperationCharacteristics:

highsolventconsumptionLow

concentrationoftheextractedproduct.Higher

extraction

yield

than

single-phase

operation48Counter-currentoperation49Residualyield：Ψn=(E-1)/(En+1-1)Extrationyield：1-Ψn=(En+1-E)/(En+1-1)《浙江工业大学学报》

,

1992

(4)

:40-57《吉林粮食高等专科学校学》

,

1997

(1)

:7-9Counter-currentoperationCharacteristics:

Oppositefeedandsolventflow;onlyonesolventisadded;Lower

solvent;Higherconcentrationofthe

product;Highestproductyield.50例：青霉素在0℃

和pH2.5时分配系数（醋酸丁酯/水）为35。用1/2体积的醋酸丁酯进行二级错流萃取和二级逆流萃取其理论收率分别是多少？(1)E=35×1/4/1=8.751-Ψ=[(8.75+1)2-1]/(8.75+1)2=98.12%(2)E=35×1/2/1=17.5n=21-Ψ=(17.52+1-17.5)/(17.52+1-1)=99.69%

错流逆流Counter-currentoperation51Example（1）如图所示是一个单级萃取装置图，现利用该装置对青霉素进行萃取。已知青霉素在0℃和pH2.5时分配系数（醋酸丁酯/水）为35。用等量的醋酸丁酯萃取料液中的青霉素和用1/3体积的醋酸丁酯萃取其萃取率分别是多少？

（2）如果要提高萃取率可以将装置改为多级萃取装置，请问目前常用的多级萃取模式是哪两种？请选择其中一种画出简易流程图并说明其主要特点。52（1）E=K×1/1＝35

1-Ψ=35/(35+1)=97.2%；E=K×1/3/1=11.67

1-Ψ=11.67/(11.67+1)=92.1%（2）多级错流萃取，多级逆流萃取。多级错流萃取特点：每级均加新鲜溶剂，故溶剂消耗量大，得到的萃取液产物平均浓度较稀，但萃取较完全。多级逆流萃取特点：料液走向和萃取剂走向相反，只加入一次萃取剂，故和错流萃取相比，萃取剂消耗少，萃取液产物平均浓度高，产物收率最高。Example5354DiscussionHow

to

further

improve

extraction

efficiency

of

artemisinin？55黄花蒿的叶粉50

g石油醚提取液2800

mL

减压旋转蒸发石油醚浸膏物质用100mL70%乙醇萃取注入萃取装置（分液漏斗）

去除蜡质乙醇萃取液100mL

用300mL含苯的石油醚萃取剂三级错流萃取萃取液300mL醇相

加5g活性炭脱色，过滤，回收溶剂浓缩液

冷却结晶粗品0.2104g青蒿素0.2254g基本工艺是：干燥→破碎→浸泡、萃取（反复进行）→浓缩提取液→粗品→精制。

减压旋转蒸发50%乙醇重结晶重复浸取四次回收，重复利用Discussion蒸发、结晶前处理青蒿素三级萃取工艺流程ExplorationQ：ThecontentofartemisinininArtemisiaannuaisrelativelylow,generallybetween0.01%and0.8%.

What

can

we

do

to

solve

this

issue?5758596061紫茴香烯青蒿酸改善细胞存活率提高产量，减少毒副产物发酵、提纯化学转化—青蒿素萃取通过合成生物学方法，改造酵母菌实现青蒿素前体的高效生产，并配套开发了将其转化为青蒿素的化学工艺62Can

you

design

a

purification

process

for

artemisinic

acid

produced

from

yeast?

ExplorationThecontentofartemisinininArtemisiaannuaisrelativelylow,generallybetween0.01%and0.8%.

What

can

we

do

to

solve

this

issue?ChemicalsynthesisGeneticbreedingSyntheticbiologyThe

separation

process

will

be

different63Exploration酵母菌体收集：将发酵液离心，收集酵母菌体。酵母菌体破碎：利用高压细胞破碎器、超声波或其他方法破碎酵母菌体，释放细胞内的artemisinicacid。萃取artemisinicacid：将破碎后的酵母菌体悬浮液与有机溶剂（如正己烷或二氯甲烷）混合，用于从悬浮液中萃取artemisinicacid。有机溶剂和水相的比例可以设置为1:1（v/v）。分离有机相：离心混合物，收集含artemisinicacid的有机相。有机相蒸发：将有机相中的溶剂蒸发掉，剩下纯化的artemisinicacid。64ExplorationTake-homemessage

in

Chinese经典育种方法：通过选择和杂交，开发出高产青蒿素的植物品种。替代育种方法：对青蒿植物进行生物技术干预，如培养基优化、生物刺激剂处理和激素处理，以提高青蒿素产量。转基因方法：通过基因工程技术，将涉及青蒿素生物合成的关键基因转移到青蒿中，以提高青蒿素的产量。合成生物学方法：通过合成生物学途径，将青蒿素生物合成途径关键酶基因转移到微生物，实现青蒿素生物合成。65Extractionequipmentsmixer-settler(混合-澄清器)extractioncolumn(柱式萃取器/微分萃取器)centrifugalseparator(离心式萃取器)66五、萃取设备混合设备：在搅拌萃取罐中加入料液和萃取溶剂，在搅拌器的作用下，二者充分混合。分离设备：用碟片式离心机将萃取相和萃余相分离（因料液和萃取溶剂不互溶，所以会形成乳浊液）。1.

单级萃取设备67主要设备-静态混合器68混合设备：静态混合器的工作原理：使流体流动冲击各种类型板元件，增加流体截面的速度梯度或形成湍流。层流时流体产生“切割-扭曲-分离-混合”运动。湍流时，流体除上述情况运动外，还会在断面方向产生剧烈的涡流，产生强烈的剪切力作用，使流体进一步分割混合，最终达到混合的目的。五、萃取设备静态混合器是常用混合器之一，与传统混合设备如搅拌器、均质管、和文氏管等相比具有结构简单，成本低、体积小，利于连续操作等优点广泛应用于化学反应、传热、乳化及萃取69静态混合器的主体组成不同型号静态混合器混合元件结构类型主要设备-静态混合器70主要设备-分离设备

达到分配平衡的两相进行分离时，可采用重力沉降法(静置分层)或离心沉降法。常用的离心沉降设备有管式离心机和碟片式离心机。71（1）脉动筛板塔（2）转盘塔在需要多级萃取的情况下,如果仍用混合器和分离机组合的方法,则设备投资过大,操作也繁琐,级数越多上述缺点越突出。这时萃取塔是最常用的设备,其中脉动塔和转盘塔应用广泛。塔式萃取操作中重相与轻相采取逆流接触的形式,搅拌脉冲为了提高液相传质设备的效率，需要外加能量，如搅拌、脉冲。五、萃取设备2.

多级萃取设备72脉动筛板塔指由于外力作用使液体在塔内产生脉冲运动的塔，轻、重液相皆可穿过塔内筛板呈逆流接触，分散相在筛板之间不凝聚分层。周期性的脉动在塔底由往复泵造成。筛板塔内加入脉动，可以增加相际接触面积及其湍动程度，故传质效率大为提高。脉动筛板的效率与脉动的振幅和频率有密切关系，若脉动过分激烈，会导致严重的轴向混合，传质效率反而降低。多级萃取设备脉动筛板塔73特点：①操作方便、密闭性好；②萃取剂用量少；③占地面积小。多级萃取设备1、脉动筛板塔74在萃取塔内安装一机械搅拌装置，促使连续相液体湍动，从而使悬浮在湍动液流中的分散相液滴被剪切成较细小的液滴，并且分布得更为均匀，萃取过程由此得到强化。对于某些分散相液滴易聚、集，或产物向萃取相转移速度较慢的物料还可以在塔板上加入填料层以进一步强化两相间物质的传递。多级萃取设备2、转盘塔75离心萃取机分为上、中、下三段，下段是第一级混合与分离区，中段是第二级，上段是第三级，每一段的下部是混合区域，中部是分离区域，上部是重相引出区域。新鲜的萃取剂由第三级加入，待萃取料液则由第一级引出。Luwesta三级离心萃取机结构多级萃取设备3、多级离心萃取机76ApplicationPurification

of

penicillin二级逆流萃取青霉素F,G,X,K,V青霉素G77C15H22O5青蒿素为无色针状晶体，熔点为156—157℃，易溶于氯仿、丙酮、乙酸乙醋和苯，可溶于乙醇、乙醚、热石油醚，微溶于冷石油醚，几乎不溶于水。由于其具有特殊的过氧基团，对热不稳定，在150℃以上分解。青蒿素是一种非常有前途的药物，被WTO称为“世界上目前唯一有效的疟疾治疗药物”。Application78一、青蒿素在黄花蒿中的含量不高，一般在0.1-0.6%，而且黄花蒿的自然资源不甚丰富二、青蒿素是细胞内产物，提取时青蒿素从细胞内释放，扩散进入提取介质比较慢，影响了提取率,增加了操作成本三、青蒿素对热不稳定,受热易分解在从植物黄花蒿中提取青蒿素的过程中，主要存在着三个问题。由于青蒿素能溶于多种有机溶剂，几乎不溶于水，因此可用有机溶剂提取植物中的有效成分，然后用柱层析或重结晶等方法分离精制得到青蒿素。青蒿素的提取Application79ConclusionKnowledge:Definition

of

extraction；Factors

influencing

extraction

efficiency；Extraction

process

operation.Ability:Design

the

extraction

process；Propose

innovativeschemesfor

improvedmaterialseparation.80ReflectionAre

there

other

strategies

to

fight

malaria？Vaccine?Can

we

purify

the

malaria

vaccine

using

extraction

method?81参见教材，写出常见的工业萃取设备种类青霉素在0℃和pH2.1时分配系数（醋酸丁酯/水）为39。用1/2体积的醋酸丁酯进行单级萃取、二级错流萃取和二级逆流萃取其理论收率分别是多少？并分析每种萃取方式的特点。根据文献内容及已学知识，设计一个完整的青蒿素前体（酵母菌生产）的多级萃取纯化工艺路线，画出萃取步骤的示意图。查阅资料，简要介绍一种目前国内的青蒿素生产工艺（请给出资料来源，或者和AI对话界面及信源截图）。Homework：82板书83萃取：青蒿素混合、分离、收集pH、温度、溶剂、盐、乳化…单级、多级错/逆流纯化工艺

Chapter7-2ReverseMicelleExtraction84ArtemisininExtraction85Introduction86黄花蒿的叶粉50

g石油醚提取液2800

mL

减压旋转蒸发石油醚浸膏物质用100mL70%乙醇萃取注入萃取装置（分液漏斗）

去除蜡质乙醇萃取液100mL

用300mL含苯的石油醚萃取剂三级错流萃取萃取液300mL醇相

加5g活性炭脱色，过滤，回收溶剂浓缩液

冷却结晶粗品0.2104g青蒿素0.2254g基本工艺是：干燥→破碎→浸泡、萃取（反复进行）→浓缩提取液→粗品→精制。

减压旋转蒸发50%乙醇重结晶重复浸取四次回收，重复利用Discussion蒸发、结晶前处理青蒿素三级萃取工艺流程Are

there

other

strategies

to

fight

malaria？Vaccine?How

to

produce,

and

most

importantly,

purify

the

malaria

vaccine?88IntroductionIntroductionInOctober2021,WHOannouncedthatchildreninmoderatetohighprevalenceareasofmalariacanreceiveRTS,S/AS01malariavaccineCan

we

use

organicsolventextractiontopurifythisvaccine?Recombinant

proteinsWe

needtoextractproteins.But?RTS

protein疟原虫的表面蛋白（CSP，环孢子表面抗原）的部分片段RTS89英国葛兰素史克ProteinsolutionInsolubleinorganicphaseProteinDenaturationQ:

Howtosolvetheproblemthattraditionalorganicsolventextractionmethodisdifficulttobeusedforproteinseparation？KeepactivitySeparationIntroduction90ThinkingWhatistheprincipleofreversedmicelleextraction?Howtousereversedmicelleextractiontopurifytherecombinantproteinvaccine?Whatarethelimitationsofreversedmicelleextraction

andwhatarethesolutions?91Reverse

Micelle

ExtractionPrincipleFormationPropertyFactorsinfluencingseparationeffectOperationprocessApplication92PrincipleHydrophilicInterfaceProteinHydrophobicSolventReverse

MicelleExtraction（RME）HydrophilicHeadHydrophobicChainSurfactantsMicelleOilyComponents93PrincipleReversedmicelles:Whensurfactantsinorganic

phaseexceedsthecriticalmicellarconcentration,theyspontaneouslyformmulti-molecularaggregateswithahydrophiliccoreandahydrophobicshell.94Reversemicelleextraction(RME):

Usereversemicellestotransferproteinsfromaqueoussolutionsintoorganicsolventswithoutdirectcontact.ProteinRME

is

an

organicsolventextraction

method.Feature:Reversemicelleextractionusessurfactantstoformreversedmicellesinanorganicphase,creatingdispersedhydrophilicmicroenvironmentswithinit.Thisenablesbiomolecules,particularlyproteins,toexistinthesehydrophilicenvironments,preventingissuesofsolubilityandirreversibledenaturationintheorganicphase.Principle9510~30

nmSurfactantsOrganic

SolventMicro-waterpoolsTheformationofreversedmicellesistheresultofself-aggregationofsurfactantmolecules.Formation961.Classification

of

reversed

micelles(a)

Singlesurfactantreversedmicellarsystems:Anionictype

(阴离子型).Representative

surfactantinreversedmicelleextractionofproteinsissodiumbis(2-ethylhexyl)sulfosuccinate

(AOT)（2-乙基己基）磺基琥珀酸钠.

Itissuitablefortheseparationofproteinswithhighisoelectricpoints.Cationictype

(阳离子型).

SuchasTOMACandCTAB.

It

issuitableforthe

proteinswithlowisoelectricpoints.Nonionicsurfactants

(非离子型).

It

canformlargerreversedmicellarsystemsandcanseparateproteinswithlargemolecularweights.Formation97AOT：AProperties：DoublechainSmallpolarheadSidechainUse

aloneLargerStableAnionictypeFormation98CTAB(溴化十六烷基三甲胺/十六烷基三甲基胺溴)－Cosurfactantarerequired.DDAB(溴化十二烷基二甲铵)CationictypeFormation99(2)

MixedsurfactantreversedmicellarsystemsItreferstosystemscomposedoftwoormoresurfactants.Generally,mixedsurfactantreversedmicelleshavehigherseparationefficiency.Formation100(1)

CriticalmicelleconcentrationTheminimumconcentrationatwhichsurfactantscanformreversedmicellesinnonpolarorganicsolventsiscalledthecriticalmicelleconcentration(CMC).TheCMCofmostsurfactantsisbetween0.1and1.0mmol/L.

Whensurfactantsformreversedmicellesinorganicsolvents,thesolubilityofwaterinorganicsolventsincreaseslinearlywithsurfactantconcentration,therefore,theCMCcanbedeterminedbymeasuringtheequilibriumwaterconcentrationinorganicsolvents.Property101(2)

Watercontentofreversedmicelles

(W0

)

W0

referstotheratioofthemolarconcentrationofwaterandsurfactantintheorganicphase.

ThelargertheW0,thelargerthereversedmicellesProperty102(3)

Solubilizingeffectofreversedmicelles水壳模型疏水区Property103DiscussionRME

can

preventissuesofsolubilityandirreversibledenaturationof

recombinant

RTS

(rRTS).104Recombinant

proteinsRTS

proteinPlease

design

a

purification

process

for

rRTS

using

RME.DiscussionPlease

design

a

purification

process

for

rRTS.

ExpressionCell

cultureCell

disruptionClarificationReversedmicellarextractionBack

extractionPurification

and

Formulation105通过质粒载体将RTS基因插入酵母表达系统离心收集细胞，超声破碎离心、0.22μm微滤去除细胞碎片反胶束萃取：水相+有机相（异辛烷）+AOT反萃取收集有机相（加入另一水相）纯化浓缩；免疫佐剂；灭菌装瓶细胞培养、诱导表达、蛋白扩增DiscussionExpressionCell

cultureCell

disruptionClarificationReversedmicellarextractionBack

extractionPurification

and

Formulation106通过质粒载体将RTS基因插入酵母表达系统离心收集细胞，超声破碎离心、0.22μm微滤去除细胞碎片反胶束萃取：水相+有机相（异辛烷）+AOT反萃取收集有机相（加入另一水相）最终纯化浓缩；免疫佐剂；灭菌装瓶细胞培养、诱导表达、蛋白扩增Howtodetail

and

optimizethe

RMEconditions?？FactorsAffectingExtraction

Efficiency1.ElectrostaticInteractions2.StericHinderanceEffects3.HydrophobicityInteractions1071.ElectrostaticInteractions(a)

pHThechargeoftheproteinrelatedtopH.

When,

pH＝pI，the

protein

is

electricallyneutral；

pH＜pI，the

proteincarriesapositivecharge；

pH＞pI，the

proteincarriesanegativecharge。FactorsAffectingExtraction

Efficiency108Forcationicsurfactants,extractionusuallyoccurswhenpH

＞pI,atwhichpointtheproteinandthepolarheadofthesurfactantinteractwitheachother.WhenpH<pI,electrostaticrepulsionwillinhibitproteinextraction.Thesituationisjusttheoppositeforthereversed-phasesystemformedbyanionicsurfactants.FactorsAffectingExtraction

Efficiency109CytochromeC:pI=10.6Nucleosidase:pI=7.8Lysozyme:pI=11.1FactorsAffectingExtraction

Efficiency110AOT

=

50

mM(b)

Ionicstrength

1.

Ions&Surfactants：Highionicstrengthreducestheelectrostaticinteractionbetweenproteinsandtheinnerwallofreversemicelles

2.

Ions&Protein：Highionicstrengthdisruptstheprotein'selectricdoublelayer,reducingsolubilityFactorsAffectingExtraction

Efficiency1112.Steric

Hinderance

Hydrophilicsubstances,suchasproteins,nucleicacids,andaminoacids,arelargemolecules.Whenthediameterofthereversephaseissmall,itwillproducespatialresistanceeffectontheprotein,thusreducingthesolubilityoftheproteininthereversephase,whichiscalledSteric

Hinderanceeffect.FactorsAffectingExtraction

Efficiency1122.Steric

Hinderance

a.

Salt

concentration：Increasingsaltconcentrationcausedehydrationofreversemicelles,reducingthe

watercontentW0

andmakingthemsmaller,whichincreasesthesterichindranceeffectanddecreasesthesolubilityofproteins.FactorsAffectingExtraction

Efficiency与蛋白质的分子量、疏水性、电荷和立体结构有关113FactorsAffectingExtraction

EfficiencyCan

we

separate

these

proteins

by

adjusting

pH

and

ionic

strength?114盐离子强度的影响

a：静电作用：1-对表面活性剂静电屏蔽；2-对蛋白双电层的破坏b：空间排阻作用：减小表面活性剂极性头之间的相互斥力，降低含水量，使反胶束变小。

这两方面的效应都会使蛋白质分子的溶解性下降，甚至使已溶解的蛋白质从反胶束中反萃取出来。

FactorsAffectingExtraction

Efficiency115b.

Molecule

weight

of

protein：Thereversemicellarextractionexperimentsattheisoelectricpointsofproteinsshowedthatthedistributioncoefficientdecreasedasthemolecularweightoftheproteinincreased,whichwasalsoduetotheincreasedsterichindrance.FactorsAffectingExtraction

Efficiency2.Steric

Hinderance

116FactorsAffectingExtraction

EfficiencyHow

to

improve

the

extraction

efficiency

of

proteins

with

large

molecular

weight？ChangingpH:ElectrostaticinteractionsV.S.sterichindrance.Increasing

theabsolutevalueof(pH-PI)如：α-糜蛋白酶(Wr25000)与牛血清蛋白(Wr68000)在中性溶液中难以通过反胶束萃取分离;但α-糜蛋白酶的萃取率在pH低于pI2-4时达到最高，而牛血清蛋白(Wr68000)在相同的系统中不发生相转移。117

c.

Surfactants

structure:

Thesmallerpolarheadsurfactantscanformreversemicelleswithalargeinternalspace,whilethelargerpolarheadsurfactantsformreversemicelleswithasmallinternalspace.2.Steric

Hinderance

FactorsAffectingExtraction

Efficiency118a.PartitionRatio：Thedistributioncoefficientofproteins

increasewithitshydrophobicity.b.SolubilizationMode：

Hydrophobicproteins

solubilizedifferentlythanhydrophilicones,likelyviamodels(2)or(4).FactorsAffectingExtraction

Efficiency3.Hydrophobicinteractions119(1)

SurfactantConcentrationIncreasingconcentrationincreasesreversemicellecountandsolubilizationcapacity.Excessivelyhighconcentrationmayformcomplexaggregatesandcomplicateback-extraction.(2)

OrganicSolventSolventtypeinfluencesreversemicellesizeandwatersolubilizationcapacity.Enables

selectivesolubilization

ofbiomoleculesbyleveragingsolvent-inducedstructuraldifferences.FactorsAffectingExtraction

Efficiency4.Other

factors120FactorsAffectingExtraction

Efficiency121(3)

TemperatureIncreasedtemperatureenhancesproteinsolubilityintheorganicphase.Higher

temperature

can

denaturize

the

proteins(4)

CosurfactantImprovesinterfacialfluidity.Facilitatesmembranecurvatureandpromotesmicelleformation.Commontypes:Medium/high-carbonalcohols,

cholesterol胆固醇,ethyleneglycol乙二醇,etc.FactorsAffectingExtraction

EfficiencyElectrostaticInteractionspHSalt

concentrationStericHinderanceEffectsSalt

concentrationMolecular

weightSurfactant

type3.HydrophobicityInteractions4.

Other

factors

(surfactant

concentration;

organic

solvent;

temperature;

co-surfactant

)122a.

Multi-stepMix-ClarificationExtractionProcess

多步间歇混合/澄清萃取过程b.

ContinuousRecyclingExtraction-StripingProcess连续循环萃取-反萃取过程Reverse

micelle

extraction

process123Can

we

separate

these

prote