Chapter

7

Extraction1IntroductionMosquitoAfrica

DiseaseHerbWhatcomestoyourmind?2IntroductionTuYouyouextracted

artemisininfromArtemisiaannua

totreatmalaria.How

to

extract

artemisinin？3ThinkingWhatisthebasicprincipleofextraction？HowtoproposearationalextractionprocessHowtoimprovetheextractionefficiencyofartemisinin？4Solventextraction

isamethodofseparationbasedonthetransferofasolutefromonesolventintoanothersolventwhenthetwosolventsarebroughtintocontact.Itisessentialthatthetwosolventsbeimmiscible不混溶的.Asolutewhichissolubleinbothphases相willdistributebetweenthetwophases兩相inadefiniteproportion特定的比例.Principle5溶質(zhì)2溶質(zhì)1原溶劑（重相）萃取劑（輕相）6PrincipleOrganicsolventextractionReversemicellesextractionAqueoustwo-phaseextractionSupercriticalfluidextraction7ClassificationofextractiontechniquesOrganicsolventextraction(有機(jī)溶劑萃取)Reversemicellesextraction(反膠束萃取)Aqueoustwo-phaseextraction(雙水相萃取)Supercriticalfluidextraction(超臨界流體萃取)8ClassificationofextractiontechniquesOrganic

Solvent

ExtractionPrincipleCharacteristicsOperationprocessFactorsinfluencingseparationeffectExtractionequipmentsApplication9DistributioncoefficientDistributionratioExtractionfactor

Extraction

efficiency

SeparationfactorDefinition10Distributioncoefficient分配系數(shù)Distributionratio分配比Extractionfactor萃取因素/萃取比Extraction

efficiency萃取率Separationfactor分離因數(shù)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=萃取相中溶質(zhì)總量/原料液中溶質(zhì)總量×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產(chǎn)/K雜Thelargertheβ,theeasieritistoseparate；Kprod=Kimp,β=1,twosubstancescannotbeseparated.Theseparationfactorvarieswiththeconcentration,composition,aqueousphasecompositionandtemperature.Theseparationfactordeterminesthepurity,andtheextractionpercentage

determinesthe

yield.23Extraction:SequenceofeventsMixingorcontactingintimatelymixingthetwosolventsuntilthesolutehasbeendistributed分配betweentheliquidsPhaseseparation相分離Collectionofseparatephases分離相的收集24Removebiochemicalsfromtheorganicphase(1)Evaporation蒸發(fā)，Crystallization結(jié)晶

fromtheorganicphase(2)Back-extraction反萃取

intoaqueousphase水相followedbyfurtherpurification純化andthencrystallization結(jié)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、前處理氯仿混合萃取離心分離兩相蒸發(fā)、干燥收集青蒿植物，粉碎，加水浸提用2倍體積的氯仿60℃萃取6小時(shí)，充分?jǐn)嚢桦x心、過濾進(jìn)行相分離，收集氯仿萃取相蒸發(fā)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時(shí)

pH=4.4不能進(jìn)行

pH>4.4青霉素由有機(jī)相轉(zhuǎn)入水相（反萃?。?/p>

pH<4.4青霉素被萃取到有機(jī)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：青霉素從水相→丁酯中，加氯化鈉，提高質(zhì)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、前處理氯仿混合萃取離心分離兩相蒸發(fā)、干燥收集青蒿植物，粉碎，加水浸提用2倍體積的氯仿60℃萃取6小時(shí)，充分?jǐn)嚢桦x心、過濾進(jìn)行相分離，收集氯仿萃取相蒸發(fā)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東晉葛洪《肘后備急方》:“青蒿一握，以水二升漬，絞取汁，盡服之”Pleasedesignapotentialextractionprocess40青蒿收集、前處理石油醚浸取乙醇萃取分相，收集蒸發(fā)、干燥收集青蒿植物，粉碎用60倍體積的石油醚30℃浸取24小時(shí)，過濾加入等體積乙醇萃取，去除葉綠素、蠟質(zhì)等蒸發(fā)干燥去除石油醚，制得青蒿素粉末Discussion分液漏斗分相，收集石油醚相如何進(jìn)一步提高萃取效率？Operation

Method（操作方式）Multi-Stage（多級(jí)萃?。〤ross-current（多級(jí)錯(cuò)流萃?。〤ounter-current（多級(jí)逆流萃?。㏒ingle

Stage（單級(jí)萃取）Extraction

operation41Extraction

operation1.

Single-stageextraction:Onlyincludesonemixerandoneseparator.1.單級(jí)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：潔霉素在20℃

和pH10.0時(shí)分配系數(shù)（丁醇/水）為18。用等量的丁醇萃取料液中的潔霉素和用1/3體積的丁醇萃取其萃取率分別是多少？(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%當(dāng)分配系數(shù)相同而萃取劑用量減少時(shí)，其萃取率下降。Singlestageoperation44Cross-currentoperation2.多級(jí)萃取—錯(cuò)流萃取45從哪里收集產(chǎn)物？經(jīng)一級(jí)萃取后，萃余率φ1：經(jīng)二級(jí)萃取后，萃余率φ2：經(jīng)n級(jí)萃取后，萃余率：Extractionyield萃取率：Cross-currentoperation46例：紅霉素在20℃

和pH9.8時(shí)分配系數(shù)（醋酸丁酯/水）為44.5。用1/2體積的醋酸丁酯進(jìn)行單級(jí)萃取和二級(jí)錯(cuò)流萃取其萃取率分別是多少？(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)《浙江工業(yè)大學(xué)學(xué)報(bào)》

,

1992

(4)

:40-57《吉林糧食高等?？茖W(xué)校學(xué)》

,

1997

(1)

:7-9Counter-currentoperationCharacteristics:

Oppositefeedandsolventflow;onlyonesolventisadded;Lower

solvent;Higherconcentrationofthe

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

和pH2.5時(shí)分配系數(shù)（醋酸丁酯/水）為35。用1/2體積的醋酸丁酯進(jìn)行二級(jí)錯(cuò)流萃取和二級(jí)逆流萃取其理論收率分別是多少？(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%

錯(cuò)流逆流Counter-currentoperation51Example（1）如圖所示是一個(gè)單級(jí)萃取裝置圖，現(xiàn)利用該裝置對(duì)青霉素進(jìn)行萃取。已知青霉素在0℃和pH2.5時(shí)分配系數(shù)（醋酸丁酯/水）為35。用等量的醋酸丁酯萃取料液中的青霉素和用1/3體積的醋酸丁酯萃取其萃取率分別是多少？

（2）如果要提高萃取率可以將裝置改為多級(jí)萃取裝置，請(qǐng)問目前常用的多級(jí)萃取模式是哪兩種？請(qǐng)選擇其中一種畫出簡易流程圖并說明其主要特點(diǎn)。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）多級(jí)錯(cuò)流萃取，多級(jí)逆流萃取。多級(jí)錯(cuò)流萃取特點(diǎn)：每級(jí)均加新鮮溶劑，故溶劑消耗量大，得到的萃取液產(chǎn)物平均濃度較稀，但萃取較完全。多級(jí)逆流萃取特點(diǎn)：料液走向和萃取劑走向相反，只加入一次萃取劑，故和錯(cuò)流萃取相比，萃取劑消耗少，萃取液產(chǎn)物平均濃度高，產(chǎn)物收率最高。Example5354DiscussionHow

to

further

improve

extraction

efficiency

of

artemisinin？55黃花蒿的葉粉50

g石油醚提取液2800

mL

減壓旋轉(zhuǎn)蒸發(fā)石油醚浸膏物質(zhì)用100mL70%乙醇萃取注入萃取裝置（分液漏斗）

去除蠟質(zhì)乙醇萃取液100mL

用300mL含苯的石油醚萃取劑三級(jí)錯(cuò)流萃取萃取液300mL醇相

加5g活性炭脫色，過濾，回收溶劑濃縮液

冷卻結(jié)晶粗品0.2104g青蒿素0.2254g基本工藝是：干燥→破碎→浸泡、萃?。ǚ磸?fù)進(jìn)行）→濃縮提取液→粗品→精制。

減壓旋轉(zhuǎn)蒸發(fā)50%乙醇重結(jié)晶重復(fù)浸取四次回收，重復(fù)利用Discussion蒸發(fā)、結(jié)晶前處理青蒿素三級(jí)萃取工藝流程ExplorationQ：ThecontentofartemisinininArtemisiaannuaisrelativelylow,generallybetween0.01%and0.8%.

What

can

we

do

to

solve

this

issue?5758596061紫茴香烯青蒿酸改善細(xì)胞存活率提高產(chǎn)量，減少毒副產(chǎn)物發(fā)酵、提純化學(xué)轉(zhuǎn)化—青蒿素萃取通過合成生物學(xué)方法，改造酵母菌實(shí)現(xiàn)青蒿素前體的高效生產(chǎn)，并配套開發(fā)了將其轉(zhuǎn)化為青蒿素的化學(xué)工藝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酵母菌體收集：將發(fā)酵液離心，收集酵母菌體。酵母菌體破碎：利用高壓細(xì)胞破碎器、超聲波或其他方法破碎酵母菌體，釋放細(xì)胞內(nèi)的artemisinicacid。萃取artemisinicacid：將破碎后的酵母菌體懸浮液與有機(jī)溶劑（如正己烷或二氯甲烷）混合，用于從懸浮液中萃取artemisinicacid。有機(jī)溶劑和水相的比例可以設(shè)置為1:1（v/v）。分離有機(jī)相：離心混合物，收集含artemisinicacid的有機(jī)相。有機(jī)相蒸發(fā)：將有機(jī)相中的溶劑蒸發(fā)掉，剩下純化的artemisinicacid。64ExplorationTake-homemessage

in

Chinese經(jīng)典育種方法：通過選擇和雜交，開發(fā)出高產(chǎn)青蒿素的植物品種。替代育種方法：對(duì)青蒿植物進(jìn)行生物技術(shù)干預(yù)，如培養(yǎng)基優(yōu)化、生物刺激劑處理和激素處理，以提高青蒿素產(chǎn)量。轉(zhuǎn)基因方法：通過基因工程技術(shù)，將涉及青蒿素生物合成的關(guān)鍵基因轉(zhuǎn)移到青蒿中，以提高青蒿素的產(chǎn)量。合成生物學(xué)方法：通過合成生物學(xué)途徑，將青蒿素生物合成途徑關(guān)鍵酶基因轉(zhuǎn)移到微生物，實(shí)現(xiàn)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(離心式萃取器)66五、萃取設(shè)備混合設(shè)備：在攪拌萃取罐中加入料液和萃取溶劑，在攪拌器的作用下，二者充分混合。分離設(shè)備：用碟片式離心機(jī)將萃取相和萃余相分離（因料液和萃取溶劑不互溶，所以會(huì)形成乳濁液）。1.

單級(jí)萃取設(shè)備67主要設(shè)備-靜態(tài)混合器68混合設(shè)備：靜態(tài)混合器的工作原理：使流體流動(dòng)沖擊各種類型板元件，增加流體截面的速度梯度或形成湍流。層流時(shí)流體產(chǎn)生“切割-扭曲-分離-混合”運(yùn)動(dòng)。湍流時(shí)，流體除上述情況運(yùn)動(dòng)外，還會(huì)在斷面方向產(chǎn)生劇烈的渦流，產(chǎn)生強(qiáng)烈的剪切力作用，使流體進(jìn)一步分割混合，最終達(dá)到混合的目的。五、萃取設(shè)備靜態(tài)混合器是常用混合器之一，與傳統(tǒng)混合設(shè)備如攪拌器、均質(zhì)管、和文氏管等相比具有結(jié)構(gòu)簡單，成本低、體積小，利于連續(xù)操作等優(yōu)點(diǎn)廣泛應(yīng)用于化學(xué)反應(yīng)、傳熱、乳化及萃取69靜態(tài)混合器的主體組成不同型號(hào)靜態(tài)混合器混合元件結(jié)構(gòu)類型主要設(shè)備-靜態(tài)混合器70主要設(shè)備-分離設(shè)備

達(dá)到分配平衡的兩相進(jìn)行分離時(shí)，可采用重力沉降法(靜置分層)或離心沉降法。常用的離心沉降設(shè)備有管式離心機(jī)和碟片式離心機(jī)。71（1）脈動(dòng)篩板塔（2）轉(zhuǎn)盤塔在需要多級(jí)萃取的情況下,如果仍用混合器和分離機(jī)組合的方法,則設(shè)備投資過大,操作也繁瑣,級(jí)數(shù)越多上述缺點(diǎn)越突出。這時(shí)萃取塔是最常用的設(shè)備,其中脈動(dòng)塔和轉(zhuǎn)盤塔應(yīng)用廣泛。塔式萃取操作中重相與輕相采取逆流接觸的形式,攪拌脈沖為了提高液相傳質(zhì)設(shè)備的效率，需要外加能量，如攪拌、脈沖。五、萃取設(shè)備2.

多級(jí)萃取設(shè)備72脈動(dòng)篩板塔指由于外力作用使液體在塔內(nèi)產(chǎn)生脈沖運(yùn)動(dòng)的塔，輕、重液相皆可穿過塔內(nèi)篩板呈逆流接觸，分散相在篩板之間不凝聚分層。周期性的脈動(dòng)在塔底由往復(fù)泵造成。篩板塔內(nèi)加入脈動(dòng)，可以增加相際接觸面積及其湍動(dòng)程度，故傳質(zhì)效率大為提高。脈動(dòng)篩板的效率與脈動(dòng)的振幅和頻率有密切關(guān)系，若脈動(dòng)過分激烈，會(huì)導(dǎo)致嚴(yán)重的軸向混合，傳質(zhì)效率反而降低。多級(jí)萃取設(shè)備脈動(dòng)篩板塔73特點(diǎn)：①操作方便、密閉性好；②萃取劑用量少；③占地面積小。多級(jí)萃取設(shè)備1、脈動(dòng)篩板塔74在萃取塔內(nèi)安裝一機(jī)械攪拌裝置，促使連續(xù)相液體湍動(dòng)，從而使懸浮在湍動(dòng)液流中的分散相液滴被剪切成較細(xì)小的液滴，并且分布得更為均勻，萃取過程由此得到強(qiáng)化。對(duì)于某些分散相液滴易聚、集，或產(chǎn)物向萃取相轉(zhuǎn)移速度較慢的物料還可以在塔板上加入填料層以進(jìn)一步強(qiáng)化兩相間物質(zhì)的傳遞。多級(jí)萃取設(shè)備2、轉(zhuǎn)盤塔75離心萃取機(jī)分為上、中、下三段，下段是第一級(jí)混合與分離區(qū)，中段是第二級(jí)，上段是第三級(jí)，每一段的下部是混合區(qū)域，中部是分離區(qū)域，上部是重相引出區(qū)域。新鮮的萃取劑由第三級(jí)加入，待萃取料液則由第一級(jí)引出。Luwesta三級(jí)離心萃取機(jī)結(jié)構(gòu)多級(jí)萃取設(shè)備3、多級(jí)離心萃取機(jī)76ApplicationPurification

of

penicillin二級(jí)逆流萃取青霉素F,G,X,K,V青霉素G77C15H22O5青蒿素為無色針狀晶體，熔點(diǎn)為156—157℃，易溶于氯仿、丙酮、乙酸乙醋和苯，可溶于乙醇、乙醚、熱石油醚，微溶于冷石油醚，幾乎不溶于水。由于其具有特殊的過氧基團(tuán)，對(duì)熱不穩(wěn)定，在150℃以上分解。青蒿素是一種非常有前途的藥物，被WTO稱為“世界上目前唯一有效的瘧疾治療藥物”。Application78一、青蒿素在黃花蒿中的含量不高，一般在0.1-0.6%，而且黃花蒿的自然資源不甚豐富二、青蒿素是細(xì)胞內(nèi)產(chǎn)物，提取時(shí)青蒿素從細(xì)胞內(nèi)釋放，擴(kuò)散進(jìn)入提取介質(zhì)比較慢，影響了提取率,增加了操作成本三、青蒿素對(duì)熱不穩(wěn)定,受熱易分解在從植物黃花蒿中提取青蒿素的過程中，主要存在著三個(gè)問題。由于青蒿素能溶于多種有機(jī)溶劑，幾乎不溶于水，因此可用有機(jī)溶劑提取植物中的有效成分，然后用柱層析或重結(jié)晶等方法分離精制得到青蒿素。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?81參見教材，寫出常見的工業(yè)萃取設(shè)備種類青霉素在0℃和pH2.1時(shí)分配系數(shù)（醋酸丁酯/水）為39。用1/2體積的醋酸丁酯進(jìn)行單級(jí)萃取、二級(jí)錯(cuò)流萃取和二級(jí)逆流萃取其理論收率分別是多少？并分析每種萃取方式的特點(diǎn)。根據(jù)文獻(xiàn)內(nèi)容及已學(xué)知識(shí)，設(shè)計(jì)一個(gè)完整的青蒿素前體（酵母菌生產(chǎn)）的多級(jí)萃取純化工藝路線，畫出萃取步驟的示意圖。查閱資料，簡要介紹一種目前國內(nèi)的青蒿素生產(chǎn)工藝（請(qǐng)給出資料來源，或者和AI對(duì)話界面及信源截圖）。Homework：82板書83萃?。呵噍锼鼗旌?、分離、收集pH、溫度、溶劑、鹽、乳化…單級(jí)、多級(jí)錯(cuò)/逆流純化工藝

Chapter7-2ReverseMicelleExtraction84ArtemisininExtraction85Introduction86黃花蒿的葉粉50

g石油醚提取液2800

mL

減壓旋轉(zhuǎn)蒸發(fā)石油醚浸膏物質(zhì)用100mL70%乙醇萃取注入萃取裝置（分液漏斗）

去除蠟質(zhì)乙醇萃取液100mL

用300mL含苯的石油醚萃取劑三級(jí)錯(cuò)流萃取萃取液300mL醇相

加5g活性炭脫色，過濾，回收溶劑濃縮液

冷卻結(jié)晶粗品0.2104g青蒿素0.2254g基本工藝是：干燥→破碎→浸泡、萃?。ǚ磸?fù)進(jìn)行）→濃縮提取液→粗品→精制。

減壓旋轉(zhuǎn)蒸發(fā)50%乙醇重結(jié)晶重復(fù)浸取四次回收，重復(fù)利用Discussion蒸發(fā)、結(jié)晶前處理青蒿素三級(jí)萃取工藝流程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瘧原蟲的表面蛋白（CSP，環(huán)孢子表面抗原）的部分片段RTS89英國葛蘭素史克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

(陰離子型).Representative

surfactantinreversedmicelleextractionofproteinsissodiumbis(2-ethylhexyl)sulfosuccinate

(AOT)（2-乙基己基）磺基琥珀酸鈉.

Itissuitablefortheseparationofproteinswithhighisoelectricpoints.Cationictype

(陽離子型).

SuchasTOMACandCTAB.

It

issuitableforthe

proteinswithlowisoelectricpoints.Nonionicsurfactants

(非離子型).

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(溴化十二烷基二甲銨)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水殼模型疏水區(qū)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通過質(zhì)粒載體將RTS基因插入酵母表達(dá)系統(tǒng)離心收集細(xì)胞，超聲破碎離心、0.22μm微濾去除細(xì)胞碎片反膠束萃取：水相+有機(jī)相（異辛烷）+AOT反萃取收集有機(jī)相（加入另一水相）純化濃縮；免疫佐劑；滅菌裝瓶細(xì)胞培養(yǎng)、誘導(dǎo)表達(dá)、蛋白擴(kuò)增DiscussionExpressionCell

cultureCell

disruptionClarificationReversedmicellarextractionBack

extractionPurification

and

Formulation106通過質(zhì)粒載體將RTS基因插入酵母表達(dá)系統(tǒng)離心收集細(xì)胞，超聲破碎離心、0.22μm微濾去除細(xì)胞碎片反膠束萃?。核?有機(jī)相（異辛烷）+AOT反萃取收集有機(jī)相（加入另一水相）最終純化濃縮；免疫佐劑；滅菌裝瓶細(xì)胞培養(yǎng)、誘導(dǎo)表達(dá)、蛋白擴(kuò)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與蛋白質(zhì)的分子量、疏水性、電荷和立體結(jié)構(gòu)有關(guān)113FactorsAffectingExtraction

EfficiencyCan

we

separate

these

proteins

by

adjusting

pH

and

ionic

strength?114鹽離子強(qiáng)度的影響

a：靜電作用：1-對(duì)表面活性劑靜電屏蔽；2-對(duì)蛋白雙電層的破壞b：空間排阻作用：減小表面活性劑極性頭之間的相互斥力，降低含水量，使反膠束變小。

這兩方面的效應(yīng)都會(huì)使蛋白質(zhì)分子的溶解性下降，甚至使已溶解的蛋白質(zhì)從反膠束中反萃取出來。

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)與牛血清蛋白(Wr68000)在中性溶液中難以通過反膠束萃取分離;但α-糜蛋白酶的萃取率在pH低于pI2-4時(shí)達(dá)到最高，而牛血清蛋白(Wr68000)在相同的系統(tǒng)中不發(fā)生相轉(zhuǎn)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膽固醇,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

多步間歇混合/澄清萃取過程b.

ContinuousRecyclingExtraction-StripingProcess連續(xù)循環(huán)萃取-反萃取過程Reverse

micelle

extraction

process123Can

we

separate

these

prote