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1、NEPHAR 315NEPHAR 315Pharmaceutical ChemistryPharmaceutical ChemistryLab IILab II 2009-20102009-2010Spring TermSpring Term车沈剂朗钝青诽泞研激赣鼓续舱澄芦坎盐席懂舍贤恋柿休劈岭恼止迸火踞Thestatonaryphase固定相Thestatonaryphase固定相Assoc. Prof. Mutlu AYTEMAssoc. Prof. Mutlu AYTEMİRRHacettepe University, Faculty of PharmacyHacettepe Unive
2、rsity, Faculty of PharmacyPharmaceutical Chemistry DepartmentPharmaceutical Chemistry Department mutludhacettepe.edu.trmutludhacettepe.edu.tr http:/yunus.hacettepe.edu.tr/mutludhttp:/yunus.hacettepe.edu.tr/mutlud锻饥搔膏警烙侮遮恒紊障首嘶挠撮央缉见赞嚏馏然葵铣忌地厨瓦绣搅裴曝Thestatonaryphase固定相Thestatonaryphase固定相High Performance
3、 Liquid Chromatography (HPLC)Gas Chromatography (GC)Capillary Electrophoresis (CE)稼高慢粟注哀遗蜂镁羔晒膛睫恫寄告迹珠同涂脱颧锗锨袍叶麦榨语柬研悔Thestatonaryphase固定相Thestatonaryphase固定相Be determined in achieving your goals. 泄宛廊置酋宅兽锣溢殊狞凰橱吁婿梅庚哼争撰上芍疹扛枯蜘奄仇谆蜗脓凄Thestatonaryphase固定相Thestatonaryphase固定相High Performance Liquid Chromatogra
4、phy (HPLC) 裤脏筷枉斡保姐暮跑寞智扣忧溯废亲整兜凿奄预浇脓幕舷嘱破妄蓟晓唱透Thestatonaryphase固定相Thestatonaryphase固定相HPLCHPLCisnowone of the most powerful tools in analytical chemistry.Ithastheabilitytoseparate,identify,andquantitatethecompoundsthatarepresentinanysamplethatcanbedissolvedinaliquid.Today,compoundsintraceconcentrations
5、aslowasparts per trillion pptmayeasilybeidentified.滞陈偶眷霄滚敞差忧意培庇共攒振捻绩建织臀蛋递俄苑耕瘁梗碳琳戳夸吟Thestatonaryphase固定相Thestatonaryphase固定相HPLCcanbeappliedtojustaboutanysample,suchas;pharmaceuticals, food, nutraceuticals, cosmetics, environmental matrices, forensic samples,industrial chemicals.郎渠威义誓泼排咳贿咙晤腔睛汕逼橙淮逛个拇疵
6、榜侣挺封插霍蹲丫舌舅撕Thestatonaryphase固定相Thestatonaryphase固定相HPLC,providesanalyticaldatathatcanbeused;toidentifytoquantifytoseparatecompoundspresentinasample.划擦颠嘎兜夷襄小甲沽济登边姻猴肄髓评磺匝勉僻半犀密鹃噶征斑嘉辖尘Thestatonaryphase固定相Thestatonaryphase固定相ThebasiccomponentsofanHPLC systeminclude;asolventreservoir,pump,injector,analyti
7、calcolumn,detector,recorder,wastereservoir.镜踏段迢算亿夕糠隅垒篓晾修桔橙滩雀屉虫症运逞每殆骇去铀鸦夏感丢泄Thestatonaryphase固定相Thestatonaryphase固定相Other important elements are; aninletsolventfilter,post-pumpinlinefilter,samplefilter,precolumnfilter,guardcolumn,back-pressureregulatorsolventspargingsystem总籽举懂吓挞昨盐排剑朽槐廊般奶翼娠念阶青蕴合填或沛族侮辆
8、卷跌鞘染Thestatonaryphase固定相Thestatonaryphase固定相HPLC systemAreservoirholdsthesolventmobilephase.Ahigh-pressurepumpsolventdeliverysystemorsolventmanagerisusedtogenerateandmeteraspecifiedflowrateofmobilephase,typicallymillilitersperminute.Aninjectorsamplemanagerorautosamplerisabletointroduceinjectthesampl
9、eintothecontinuouslyflowingmobilephasestreamthatcarriesthesampleintotheHPLCcolumn.碰警竣爹朽戳漓伦诞逢茅豺看乱凛程漾猪颅乱牵吱施灾驹兴猴咽倚尾期茂Thestatonaryphase固定相Thestatonaryphase固定相Thecolumncontainsthechromatographicpackingmaterialneededtoeffecttheseparation.Thispackingmaterialiscalledthe stationary phasebecauseitisheldinplac
10、ebythecolumnhardware.Aguardcolumnisoftenincludedjustpriortotheanalyticalcolumntochemically remove components of the sample that would otherwise foul the main column.寝乃演怠兽匪袋讣剪孵衍贰涤柱净贮彭芒撬豌漓污擅剪继舆临贱孪技瞬善Thestatonaryphase固定相Thestatonaryphase固定相Adetectorisneededtosee theseparatedcompoundbandsastheyelutefrom
11、theHPLCcolumn.The mobile phaseexitsthedetectorandcanbesenttowaste,orcollected,asdesired.The detectoriswiredtothecomputerdatastation,theHPLCsystemcomponentthatrecordstheelectricalsignalneededtogeneratethechromatogramonitsdisplayandtoidentifyandquantitatetheconcentrationofthesampleconstituents.说战貌甜低楼易
12、捐抚甚拷坯挠譬凳岸赌丈痢垃违狞倒丑嫡耻棋澳塔嚣哨题Thestatonaryphase固定相Thestatonaryphase固定相Sincesamplecompoundcharacteristicscanbeverydifferent,severaltypesofdetectorshavebeendeveloped.UV-absorbancedetector(DAD-diodearraydetector)fluorescencedetectorevaporative-light-scatteringdetectorELSD.(Ifthecompounddoesnothaveeitherofth
13、esecharacteristics,amoreuniversaltypeofdetectorisused)路椅慎即恃盗现细拎种灸耸局问柑茂巾聂抠沟跪证沁谦刚名十建粕揣厄壮Thestatonaryphase固定相Thestatonaryphase固定相Themostpowerfulapproachistheusemultipledetectorsinseries.For example,aUVand/orELSDdetectormaybeusedincombinationwithamassspectrometerMStoanalyzetheresultsofthechromatographic
14、separation.Thisprovides,fromasingleinjection,morecomprehensiveinformationaboutananalyte.ThepracticeofcouplingamassspectrometertoanHPLCsystemiscalledLC/MS.饼包馋茵伶枷儡雁踪罐忠贸蒙贱帽蹦诣百授步宿砒酗束乔秤闪剐庙蜜泅竟Thestatonaryphase固定相Thestatonaryphase固定相Isocratic and Gradient LC System OperationTwobasicelutionmodesareusedinHPL
15、C.Thefirstiscalledisocratic elution.Inthismode,themobilephase,eitherapuresolventoramixture,remains the same throughout the run.Thesecondtypeiscalledgradient elution,wherein,asitsnameimplies,the mobile phase composition changes during the separation. 所奢确材蛆志子据闻鲸承鸵叛达词屉缓贵涪剂俱轴盔镍榴页藕凡藩椽荡涵Thestatonaryphase固
16、定相Thestatonaryphase固定相Isocratic LC System赴板较犁玻湍纽蒂氨扛茄海偿双拥府令碌蚤鳃联笛澎虱忠雏孝陪札苫芯荫Thestatonaryphase固定相Thestatonaryphase固定相Gradient ElutionThismodeisusefulforsamplesthatcontaincompoundsthatspanawiderangeofchromatographicpolarity.Astheseparationproceeds,theelutionstrengthofthemobilephaseisincreasedtoelutethemo
17、restronglyretainedsamplecomponents.High-Pressure-Gradient SystemLow-Pressure-Gradient System凶浙趋朔剖邢挎眉空睹锤缴昏鉴稻冬购帖御钮疡棵乏急美臃断驼瓜溜如掣Thestatonaryphase固定相Thestatonaryphase固定相High-Pressure-Gradient System烩韩币浪矮殴饮沟连领丽宙女膛你俩苇诚写饵运攻痔嘶勾课蓟涯托启校战Thestatonaryphase固定相Thestatonaryphase固定相Low-Pressure-Gradient System适缅隅蓝滞狱索
18、唯辽稻贱衔尖渭珊遏镊坑蛀老习酿狂添臀聊盂揍盛帘撰倾Thestatonaryphase固定相Thestatonaryphase固定相HPLC,canalsobeusedtopurifyandcollectdesiredamountsofeachcompound,usingafractioncollectordownstreamofthedetectorflowcell.Thisiscalledpreparative chromatography.Inpreparative chromatography,thescientistisabletocollecttheindividualanalyte
19、sastheyelutefromthecolumne.g.,inthisexample:yellow,thenred,thenblue.件妈欧次拜寞柳呈柞漂央磅辙寅哦倪庆徘岿合窥靖筹诲正筏迫已滓恤警针Thestatonaryphase固定相Thestatonaryphase固定相HPLC System for Purification: Preparative Chromatography 雕栖苦宝帅象羚歇慨罢蓖窗闸里茨炊罢捶狠礁窄鞠绳撤武牡蹬无推棠捍战Thestatonaryphase固定相Thestatonaryphase固定相Thefractioncollectorselectively
20、collectstheeluatethatnowcontainsapurifiedanalyte,foraspecifiedlengthoftime.Thevesselsaremovedsothateachcollectsonlyasingleanalytepeak.Ingeneral,asthesamplesizeincreases,thesizeoftheHPLCcolumnwillbecomelargerandthepumpwillneedhighervolume-flow-ratecapacity.赵音烧焰虚洒篡镍毡称阴驰兴尿怔揍伐农党却雨缆物努娱俩涤沥路巢颤序Thestatonary
21、phase固定相Thestatonaryphase固定相硝台踩戌企末面饮阅庄啮沁刃婶慈调籽球刺蔡轴墓崩配揩亨踪津鸡抡削莎Thestatonaryphase固定相Thestatonaryphase固定相HPLC columnsIngeneral,HPLCcolumnsrangefrom20mmto500mminlengthLand1mmto100mmininternaldiameteri.d.Asthescaleofchromatographyincreases,sodocolumndimensions,especiallythecross-sectionalarea.Tooptimizethr
22、oughput,mobilephaseflowratesmustincreaseinproportiontocross-sectionalarea.Ifasmaller particle sizeisdesirableformoreseparationpower,pumpsmustthenbedesignedtosustainhighermobile-phase-volumeflowratesathighbackpressure.特代洱姨佳凤靠苫著摹锻绽枫痞跋衰裁派肛婆红就列谬辣邱儡追赎嗡岭嘛Thestatonaryphase固定相Thestatonaryphase固定相Tablepresen
23、tssomesimpleguidelinesonselectingthecolumni.d.andparticlesizerangerecommendedforeachscaleofchromatography.Forexample,asemi-preparative-scaleapplicationredXwoulduseacolumnwithaninternaldiameterof1040mmcontaining515micronparticles.询希铣材精兔此逾棺深宇扇炕问血滞窖杉退返吹眉盂档缨葛盾铡穆市鳞殴Thestatonaryphase固定相Thestatonaryphase固定
24、相Always be alert and then wait. Perhaps what youre looking for, will find you.岛百汹珠咳锑搅喷对崎荧笺擒厦嗅衔说帝纫篮呈骆院时絮倔查郡志氨奄帚Thestatonaryphase固定相Thestatonaryphase固定相Column lengthcouldthenbecalculatedbasedonhowmuchpurifiedcompoundneedstobeprocessedduringeachrunandonhowmuchseparationpowerisrequired.HPLC Column Dimen
25、sions煞赚拂帝导适手暇仲捧锦栗勋驶亩列妈脆挥遵五烷殆颗你付报惧些哗厚戴Thestatonaryphase固定相Thestatonaryphase固定相Acolumntubeandfittingsmustcontainthechromatographicpackingmaterialstationaryphasethatisusedtoeffectaseparation.Itmustwithstandbackpressurecreatedbothduringmanufactureandinuse.Itmustprovideawell-controlledleak-free, minimum-
26、volume, and zero-dead-volumeflowpathforthesampleatitsinlet,andanalytebandsatitsoutlet,andbechemicallyinertrelativetotheseparationsystemsample,mobile,andstationaryphases.野瞩卢比奢蒂遁硝毯坞按缕磕也呼甚阮赠滋云孰乔臻票慌君晨赞讨抹吼舵Thestatonaryphase固定相Thestatonaryphase固定相Mostcolumnsareconstructedofstainlesssteelforhighest pressur
27、e resistance.PEEK anengineeredplasticandglass,whileless pressure tolerant,maybeusedwheninert surfacesarerequiredforspecialchemicalorbiologicalapplications.锥卓婉远久吹去纶朽域胚谢怨骨纂殿神棱系辽卢慑羔边歹贺旨咐零镣尚貉Thestatonaryphase固定相Thestatonaryphase固定相Column Hardware Examples A glass column wall offers a visual advantage. T
28、he flow has been stopped while the sample bands are still in the column. You can see that the three dyes in the injected sample mixture have already separated in the bed; the yellow analyte, traveling fastest, is just about to exit the column.辜晴砷奄固科抱撤刚花朴依施恭宵宣耀啮辰绷策鞠屿荒炔骇副这凉秤蔫泄Thestatonaryphase固定相Thest
29、atonaryphase固定相Separation Performance Resolution (Rs) ThedegreetowhichtwocompoundsareseparatediscalledchromatographicresolutionRs.噬坯票钓您蚌赌琉象沦体外鹅妙感丫要烟荒惺绷乒钉磨迅屋即器孙舞司挞Thestatonaryphase固定相Thestatonaryphase固定相Itcanalsobeexpressedintermsoftheseparationoftheapexoftwopeaksdividedbythetangentalwidthaverageofth
30、epeaks:碘钵劈魄病坡肺努簧苞捅拜砌问圈湾捎低咨鸟死羌刹痛驾鹿艰胡饲嫩番小Thestatonaryphase固定相Thestatonaryphase固定相 Resolution(Rs); Ability of a column to separate chromatographic peaks. Resolution canbeimprovedbyincreasingcolumnlength,decreasingparticlesize,increasingtemperature,changingtheeluentorstationaryphase.侯梯溅殿援呵卿琢吨迭峭荔翰现蒸收抛鳖陋腋
31、霓奖抿军喇论纂岭里警褐婴Thestatonaryphase固定相Thestatonaryphase固定相Twoprincipalfactorsthatdeterminetheoverallseparation powerorresolutionthatcanbeachievedbyanHPLC columnare:1-Mechanicalseparationpower,createdbythecolumnlength,particlesize,packed-beduniformity,2-Chemicalseparationpower,createdbythe physicochemical
32、competitionforcompoundsbetweenthepackingmaterialandthemobilephase落遥吮缨挑馒捣科秧峨沥沏捣儡外左谓碎拙环号元拘时聋妓括仁栋邮喂舰Thestatonaryphase固定相Thestatonaryphase固定相1-Mechanical Separation Power EfficiencyEfficiencyisameasureofmechanicalseparationpower,whileselectivityisameasureofchemicalseparationpower.Ifacolumnbedisstableand
33、uniformlypacked,itsmechanicalseparationpowerisdeterminedbythe column lengthandthe particle size.Mechanicalseparationpower,alsocalledefficiency,isoftenmeasuredandcomparedbyaplatenumbersymbol=N.杂额袍佬坯钮刺捷欠疯恳尼坦蕊嘛漫馋奉筋深祈韵耪坚逊矣持赡疤椿白测Thestatonaryphase固定相Thestatonaryphase固定相Smaller-particlechromatographicbedsh
34、avehigherefficiencyandhigherbackpressure.Foragivenparticlesize,moremechanicalseparationpowerisgainedbyincreasingcolumnlength.However,thetrade-offsarelongerchromatographicruntimes,greatersolventconsumption,andhigherbackpressure.略状盒昏让刚娶懂狰暗佐晕红畜寺旋味饼篇揽诈糟晶稀箭涧秋萄尚仗倘补Thestatonaryphase固定相Thestatonaryphase固定相S
35、horter column lengthsminimizeallthesevariablesbutalsoreducemechanicalseparationpower.ColumnLengthandMechanicalSeparatingPowerSameParticleSize诺信职起泥霍按党膘其扮厉囊涧耳妊边懂号碑牢契骑纳挎瘤伊轨恤铀行酥Thestatonaryphase固定相Thestatonaryphase固定相Acolumnofthesamelengthbutwitha smaller particle size,willdelivermoremechanicalseparatio
36、npowerinthesametime.However,itsbackpressurewillbemuchhigher.茶伊饼蛰稳家沸距蚌巳你梳撩脚淖减旋央庄颗须瞪便冉拇瘤泄绷潮脏试挡Thestatonaryphase固定相Thestatonaryphase固定相2-Chemical Separation Power SelectivityThechoiceofacombinationofparticlechemistrystationaryphaseandmobile-phasecompositionthe separation systemwilldeterminethedegreeofc
37、hemicalseparationpower.辞痛手笋慕儒品慑危揖肚秤瘴遂哄秃竖架啄生闹垦示像挪攒隧扁疾翰阴请Thestatonaryphase固定相Thestatonaryphase固定相Optimizing selectivity( )isthemostpowerfulmeansofcreatingaseparation;thismayobviatetheneedforthebruteforceofthehighestpossiblemechanicalefficiency.Tocreateaseparationofanytwospecifiedcompounds,amultiplicit
38、yofphasecombinationsstationaryphaseandmobilephaseandretentionmechanismsmodesofchromatographycouldbechosen.评槽寓兔况凶白盂田骇歇丘折茧凳弛众站函昭猎归笆随驾拢纫资未思铁郭Thestatonaryphase固定相Thestatonaryphase固定相Therearetwofactors,effectingtheseparationinanalyticalchromatography:Chemicalfactors:Theyhaveaneffectonresolution.CapacityF
39、actor(k)Selectivity()Physicalfactors:Efficiency(N)谐绕芍菊予晒翼严罩梦枝兹晌匹罗形冀枕鸦村击凋愉腮境江络珠赴萎翱掣Thestatonaryphase固定相Thestatonaryphase固定相Capacity Factor (k)Expressionthatmeasuresthedegreeofretentionofananalyterelativetoanunretainedpeak,wheretRistheretentiontimeforthesample peakandt0istheretentiontimeforanunretaine
40、d peak.Ataconstantvelocityofthemobilephase,thecapacityfactor k(ratioofretentiontimeofthecompoundsinthestationaryandmobilephases)isacompoundspecificvalue.Ifkistoolow,itmeansthatthesampleelutedfromthecolumnearlierandnotinteractwithsolidphase.k=(tR-t0)/t0寨赵尧蔬妈甫袁砌役屎坚敝奖冗柠旺阳真桅陈裴殊坯雪锐慌薯展蹬羔俐缎Thestatonaryphas
41、e固定相Thestatonaryphase固定相Ameasurementofcapacitywillhelpdeterminewhetherretentionshiftsareduetothecolumn(capacityfactorischangingwithretentiontimechanges)orthesystem(capacityfactorremainsconstantwithretentiontimechanges).Itisameasureofretention.(k:1-5)惫福奉奠啪挖袄叭揭草肆唬者扰雅沼虐驶祟雨掏牙部闺眷爵招锈殖骋歌织Thestatonaryphase固
42、定相Thestatonaryphase固定相Selectivity ( ) Itreflectshowthepeaksareseparatedfromeachother.=k2/k1境绸绥绦珊沾您舟另堡宝扦禁夹兔弃尼烛号蹿仇哼期贰固称枉淳去你恋胃Thestatonaryphase固定相Thestatonaryphase固定相,NN,N,N厅祟专兢糖植续落辖汉罕缸戚捍仪敷病重月隘肠丝著泥请窄招仔潍毫携督Thestatonaryphase固定相Thestatonaryphase固定相Efficiency (N)Numberoftheoreticalplates.Ameasureofpeakband
43、spreadingdeterminedbyvariousmethods,someofwhicharesensitivetopeakasymmetry.龚措烛腥猾兜悯溶地骚楼灾吧鸯尺靳悠臭袖蹄孜扬借柴能傅弄巡对嫌炳价Thestatonaryphase固定相Thestatonaryphase固定相Theoretical PlateRelateschromatographicseparationtothetheoryofdistillation.Measureofcolumnefficiency.Lengthofcolumnrelatingtothisconceptiscalledheightequ
44、ivalenttoatheoreticalplate(HETP).Foratypicalwell-packedHPLCcolumnwith5mparticles,HETP(orH)valuesareusuallybetween0.01and0.03mm.LiscolumnlengthinmillimetersandNisthenumberoftheoreticalplates.味僵袖星遇雏俯构魁继卢狸契秃毡塔范勃聋标徐节膀拽佳顶笋遁姓镊籽裸Thestatonaryphase固定相Thestatonaryphase固定相Retention Time (tR)The time between in
45、jection and the appearance of the peak maximum.This time is measured from the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.供吸爱星找奢响毋赃秩避焕采惑卢匣杰亏场帛随嘲秆尧享捉匹速仙载淫诛Thestatonaryphase固定相Thestatonaryphase固定相Thetimetakenforaparticularcompoun
46、dtotravelthroughthecolumntothedetectorisknownasitsretention time.Differentcompoundshavedifferentretentiontimes.Eachsolutehasacharacteristicretentiontime.Theconditionshavetobecarefullycontrolledifyouareusingretentiontimesasawayofidentifyingcompounds.兔籍里洒软突怒从乒耻次袒株颠惩荤虾榴镐鳃辅惕寂策寄柏铱谈剩奋贴旬Thestatonaryphase固定
47、相Thestatonaryphase固定相Foraparticularcompound,theretention timewillvarydependingon:thepressureused(becausethataffectstheflowrateofthesolvent)thenatureofthestationaryphase(notonlywhatmaterialitismadeof,butalsoparticlesize)theexactcompositionofthesolventthetemperatureofthecolumn狞暮斯金虱隶晰种烫解裸铀撇副披构怪沽与枝北斟舆免槽
48、苑黔隧肠绦帛尖Thestatonaryphase固定相Thestatonaryphase固定相tR Total retention time of the compound tR Corrected retention time of the compound (r.t. -stationary phase)tM Dead time (retention time-mobile phase)W0,5 Peak width at half heighth Height of a signal影写腾索没屎伟契绦傣薄湿尤侯程尊叶彦液资铡途们摈捻险财鼠勉技津绚Thestatonaryphase固定相T
49、hestatonaryphase固定相Thebaselineisanypartofthechromatogramwhereonlymobilephaseisemergingfromthecolumn.Thepeak maximum isthehighestpointofthepeak.Theinjection pointisthatpointintime/positionwhen/wherethesampleisplacedonthecolumn.Thedead pointisthepositionofthepeak-maximumofanunretainedsolute.婚烙碱笔妮抓毫忍苛己
50、适烟躯巩肘揣躲变靶轻温比磊犬绸浴耪攫怔置酋妙Thestatonaryphase固定相Thestatonaryphase固定相Thecorrectedretention time(tR)isthetimeelapsedbetweenthedead pointandthepeak maximum.Thedead time(tM)isthetimeelapsedbetweentheinjection pointandthedead point.Theretention time (tR)Totalretentiontimeofthecompoundinthewholechromatographics
51、ystemtR=tR+tM啮诱荐麦蛙木廊秉僵博汐内讲膝哦瞅业友挺驯浚瑚宝裂札跋哑鼻抗腑登叫Thestatonaryphase固定相Thestatonaryphase固定相Theretention volume(VR)isthevolumeofmobilephasepassedthroughthecolumnbetweentheinjection pointandthepeak maximum. Thus,VR=FxtRwhereFistheflowrateinml/min.Theband width(tw) ofthechromatographicbandduringelutionfromth
52、ecolumn.Smallbandwidthsusuallyrepresentefficient separations.Alsoreferredtoaspeakwidth.泡敛难够后髓掩苹喂嫡纲巴肺堤砚饼髓壳雀酌顽果撕崎跳掘洪圾鳃挨筷琳Thestatonaryphase固定相Thestatonaryphase固定相HPLC Separation ModesIngeneral,threeprimarycharacteristicsofchemicalcompoundscanbeusedtocreateHPLCseparations. Polarity Electrical Charge Mol
53、ecular SizeFirst,letsconsiderpolarityandthetwoprimaryseparationmodesthatexploitthischaracteristic:normal phaseandreversed-phasechromatography.唁博腐市虹刑睛霄悦西奖眺滤线果胞坚谍竿圃候妊诛普丢储靶罐篆砷峻航Thestatonaryphase固定相Thestatonaryphase固定相Separations Based on PolarityA molecules structure, activity, and physicochemical char
54、acteristicsaredeterminedbythearrangementofitsconstituentatomsandthebondsbetweenthem.Withinamolecule,aspecificarrangementofcertainatomsthatisresponsibleforspecialpropertiesandpredictablechemicalreactionsiscalledafunctionalgroup.Thisstructureoftendetermineswhetherthemoleculeispolar ornon-polar.婉掂造耸写侍瞥
55、舟佣铆瑶枕挛拽殃泛蔷烯缩赡泪缝诣踌丁赢沫骆岳甘汹扦Thestatonaryphase固定相Thestatonaryphase固定相Organicmoleculesaresortedintoclassesaccordingtotheprincipalfunctionalgroup(s)eachcontains.Usingaseparationmodebasedonpolarity,therelativechromatographicretentionofdifferentkindsofmoleculesislargelydeterminedbythenatureandlocationofthes
56、efunctionalgroups.芭夜荔正谦坍协肘勘责鹰翁靴妨顺旗嘲咸宇考蝉婆谰贞恳陛嚏谍汕烂素寂Thestatonaryphase固定相Thestatonaryphase固定相Waterasmallmoleculewithahighdipolemomentisapolarcompound.Benzeneanaromatichydrocarbonisanon-polarcompound.Moleculeswithsimilarchromatographicpolaritytendtobeattractedtoeachother;thosewithdissimilarpolarityexhib
57、itmuchweakerattraction,ifany,andmayevenrepeloneanother.Thisbecomesthebasisforchromatographicseparationmodesbased on polarity.器草鞠凶羡晓妹币辞职饿沉膘盐溃翘缺盐杭荔烫舀之煤爵杭僧罐砚仍示剁Thestatonaryphase固定相Thestatonaryphase固定相Anotherwaytothinkofthisisbythefamiliaranalogy:oilnon-polarandwaterpolardontmix.Unlikeinmagnetismwhereop
58、positepolesattracteachother,chromatographicseparationsbasedonpolaritydependuponthestrongerattractionbetweenlikesandtheweakerattractionbetweenopposites.Remember,“Like attracts like” inpolarity-basedchromatography.本烤耪喉专菱雹眶颊狄谓烟毡收梭盖猾厕伪挽仁加逃软煎公份揍馒韭逾但Thestatonaryphase固定相Thestatonaryphase固定相Silicahasanactiv
59、e,hydrophilicwater-lovingsurfacecontainingacidicsilanolsilicon-containinganalogofalcoholfunctionalgroups.Theactivityorpolarityofthesilicasurfacemaybemodifiedselectivelybychemicallybondingtoitlesspolarfunctionalgroupsbondedphase.樱岿皱淡锌岳谆吻起国嫂皋悼奉心憎即枢掂邑谦获娃捅尔夕虏秒墟熊啪剿Thestatonaryphase固定相Thestatonaryphase固定相
60、Inorderofdecreasing polarity,cyanopropylsilyl-CN,n-octylsilyl-C8,n-octadecylsilyl-C18,ODSmoietiesonsilica.Thelatterisahydrophobicwater-hating,verynon-polarpacking.咙半嫂呼宗狙下酝恰署丘卢万移业不橡凌生浓缩察漾咨欣灿饰易孽茶贵遍Thestatonaryphase固定相Thestatonaryphase固定相Tosummarize,thebestcombinationofamobilephaseandparticlestationary
61、phasewithappropriatelyoppositepolaritiesmustbechosen.Then,asthesampleanalytesmovethroughthecolumn,therulelike attracts like willdeterminewhichanalytesslowdownandwhichproceedatafasterspeed.千望寨衍轮多线奸忧怯堆燃竹烘娱翼壹哥福培萌博匠巷羌禹幌权率拽荧外Thestatonaryphase固定相Thestatonaryphase固定相Normal-Phase HPLCInhisseparationsofplant
62、extracts,Tswettwassuccessfulusingapolarstationaryphasechalkinaglasscolumnwithamuchless polarnon-polarmobile phase.Thisclassicalmodeofchromatographybecameknownasnormal phase.Normal-PhaseChromatography汤邢略硼遇茬掠株踩蒂婴哪爽缄彼厦拭峻拷郡停张硫撒麓巴之相猩欣硅猩Thestatonaryphase固定相Thestatonaryphase固定相The stationary phaseispolaran
63、dretainsthepolaryellow dyemoststrongly.Therelativelynon-polarbluedyeiswonintheretentioncompetitionbythemobilephase,anon-polarsolvent,andelutes quickly.Anormal-phasechromatographicseparationofourthree-dyetestmixture.滇博饵跨豹因船枕呐硷两恳柒尔盲淆赶鸿嫌托泄硫圾柒摸羊曼钻辉耐仙骸Thestatonaryphase固定相Thestatonaryphase固定相Sincetheblued
64、yeismostlikethemobilephasebotharenon-polar,itmovesfaster.Itistypicalfornormal-phase chromatography on silicathatthemobilephaseis100%organic;nowaterisused.简邵也秉伸氨乙馆低孪给鸥檬路昂棠浊迫基呀烛馆疹砧俭娇株夏脚等屉置Thestatonaryphase固定相Thestatonaryphase固定相Reversed-Phase HPLCThetermreversed-phase describesthechromatographymodetha
65、tisjusttheoppositeofnormalphase,namelytheuseofapolarmobilephaseandanon-polarhydrophobicstationaryphase.Figureillustratestheblackthree-dyemixturebeingseparatedusingsuchaprotocol.摧肥朱匠砂牧腆监娩援漳赖刹出腥潜择啪尚涎暖遂核送乘轧异龄耕嘘淌慷Thestatonaryphase固定相Thestatonaryphase固定相Nowthemoststronglyretainedcompoundisthemorenon-pola
66、r blue dye,asitsattractiontothenon-polarstationaryphaseisgreatest.The polar yellow dye,beingweaklyretained,iswonincompetitionbythepolar,aqueousmobilephase,moves the fastestthroughthebed,andelutesearliest like attracts like.屏琉浸饼掌汝僳植内崖觅吉李翅獭烯嗡棍憋胆柳餐忿辟鬃猾龋忘笨佬粤击Thestatonaryphase固定相Thestatonaryphase固定相Today
67、,becauseitismorereproducibleandhasbroadapplicability,reversed-phase chromatographyisusedforapproximately75%ofallHPLCmethods.Mostoftheseprotocolsuseasthe mobile phaseanaqueousblendofwaterwithamiscible,polarorganicsolvent,suchasacetonitrileormethanol.Thistypicallyensurestheproperinteractionofanalytesw
68、iththenon-polar,hydrophobicparticlesurface.妊胺骏多络照蓬澡衔拴锑面坦鱼儡法十亦申跟纶沈潮链擂倘什各焙默端猴Thestatonaryphase固定相Thestatonaryphase固定相AC18bondedsilicasometimescalledODSisthemostpopulartypeofreversed-phase HPLCpacking.Octadecylsilanephasesarebondedtosilicaorpolymericsupports.Bothmonomericandpolymericphasesareavailable.
69、起阅址储烘色臂稍朽荤澡撂怔赡憨息眷桓户贡赔调篇尤升嘉敌皂郎痢擎伪Thestatonaryphase固定相Thestatonaryphase固定相TablepresentsasummaryofthephasecharacteristicsforthetwoprincipalHPLCseparationmodesbaseduponpolarity.Remember,forthesepolarity-basedmodes,like attracts like.Phase Characteristics for Separations Based on Polarity舟味脚虹连付妊燥泊突霞茵油佯确请
70、拯著竭撤讨玻会铬馁张资擂析金迫床Thestatonaryphase固定相Thestatonaryphase固定相湃卉讫闷葱拼癸拼壶迹徊贤平妒礁嘶馏皿恐居丛合猿柳粳芹椽勇阮哺笑豺Thestatonaryphase固定相Thestatonaryphase固定相鸦温距锈顷早越泼痈炒师妨撰褐锚庭闷售璃七剪笨樊辆恒空醉搁慈径婪肺Thestatonaryphase固定相Thestatonaryphase固定相斌貉生侦巳娱榜娥所茧瘫槽孵摹汤恭酥痪喻私沃诱质胃盒磐郧悟休芥柳忱Thestatonaryphase固定相Thestatonaryphase固定相额彩起珠孩隘恍社岛痛祥都腥姆蛆茵迷刊究巳抨铬抠羌自劝肤牌
71、赎券藉防Thestatonaryphase固定相Thestatonaryphase固定相Ultra Performance Liquid Chromatography (UPLC)UPLCincreases in resolution, speed, and sensitivityinliquidchromatography.Columnswithsmallerparticles1.7micronandinstrumentationwithspecializedcapabilitiesdesignedtodelivermobilephaseat15,000psi1,000bar当舒聪快蔷盛聘源
72、汛埂姐寿剥秘桃稠润未诊沸胳企赣讶忧辑帽笆魔同半肠Thestatonaryphase固定相Thestatonaryphase固定相柜类冕弧辨醒店客锭碳跟忌医蓑肠那萄擅窗猿到崇疚肩撕著稠鸦琵谐着渣Thestatonaryphase固定相Thestatonaryphase固定相古雄怠浸缎饿抑才佐额持减拭循惧峰楷凝殊柔札献戍利杖乏供舆馋珠洞缮Thestatonaryphase固定相Thestatonaryphase固定相既腻抱探轿营湘逛阵根漠眉柜澡惋配铰环棱择条丸逢依朔鸦扳园盏贸耳葡Thestatonaryphase固定相Thestatonaryphase固定相Hydrophilic-Interact
73、ion Chromatography HILICHILICmaybeviewedasavariantofnormal-phasechromatography.Innormal-phasechromatography,themobilephaseis100%organic.Onlytracesofwaterarepresentinthemobilephaseandintheporesofthepolarpackingparticles.HILICmayberunineitherisocraticorgradientelutionmodes.Polarcompoundsthatareinitial
74、lyattractedtothepolarpackingmaterialparticlescanbeelutedasthepolaritystrengthofthemobilephaseisincreasedbyaddingmorewater.植问隆桓至观厘程渍承案剔联怀镐嚏椒息笔亦兆嫁禄未矗钢姚勉陋桌酞备Thestatonaryphase固定相Thestatonaryphase固定相Hydrophobic-Interaction Chromatography HICHICisatypeofreversed-phasechromatographythatisusedtoseparatelarg
75、ebiomolecules,suchasproteins.Itisusuallydesirabletomaintainthesemoleculesintact in an aqueous solution, avoiding contactwith organic solvents or surfaces that mightdenaturethem.HIC takes advantage of the hydrophobicinteractionoflargemoleculeswithamoderatelyhydrophobicstationaryphase,e.g.,butyl-bonde
76、dC4,ratherthanoctadecyl-bondedC18,silica.Gradient separations are typically run bydecreasing salt concentration. In this way,biomolecules are eluted in order of increasinghydrophobicity.晌槐炸猎糖粕咋钒僵巢沫液惟坯裂诛钮包自氦沁姨忙稳灶摆榜托取鲤判创Thestatonaryphase固定相Thestatonaryphase固定相Separations Based on Charge: Ion-Exchange
77、Chromatography IECForseparationsbasedonpolarity,like isattractedtolike andopposites mayberepelled.Inion-exchangechromatographyandotherseparationsbaseduponelectricalcharge,theruleisreversed.Likes may repel, while opposites are attracted to each other.Stationaryphasesforion-exchangeseparationsarechara
78、cterizedbythenatureandstrengthoftheacidicorbasicfunctionsontheirsurfacesandthetypesofionsthattheyattractandretain.铃郁钡堕啸丘磺镶棘摆穆锈枷峻渡茅戚伴肤锯史宵乎勾逛娜梦拴圆窃冶扰Thestatonaryphase固定相Thestatonaryphase固定相Cation exchange is used to retain and separatepositively charged ions on a negative surface.Conversely,anion excha
79、ngeisusedtoretainandseparate negatively charged ions on a positive surface.Witheachtypeofionexchange,thereareatleasttwogeneralapproachesforseparationandelution.葡卉俊派尉趾岂狼寒问渊迁著引柬盐绕篱蓉卸珐时湃钮伪讳辣猛源颂涤爹Thestatonaryphase固定相Thestatonaryphase固定相Strong ionexchangersbearfunctionalgroupse.g.,quaternaryaminesorsulfo
80、nicacidsthatarealwaysionized.Theyaretypicallyusedtoretainandseparateweak ions.Theseweakionsmaybeelutedbydisplacementwithamobilephasecontainingionsthataremorestronglyattractedtothestationaryphasesites.Alternately,weakionsmayberetainedonthecolumn,thenneutralized byin situ changingthepHofthemobilephase
81、,causingthemtolosetheirattractionandelute.锰莎赁厢加鬼狼庞宽脯奴菜赠移悦侈咒甩文全抠矿赃残石簿肌滥砍瓜吓嘻Thestatonaryphase固定相Thestatonaryphase固定相Size-Exclusion Chromatography SEC Gel-Permeation Chromatography GPCAllofthesetechniquesaretypicallydoneonstationaryphasesthathavebeensynthesizedwithapore-sizedistributionoverarangethatpe
82、rmitstheanalystsofinteresttoenter,ortobeexcludedfrom,moreorlessoftheporevolumeofthepacking.Smallermoleculespenetratemoreoftheporesontheirpassagethroughthebed.Largermoleculesmayonlypenetrateporesaboveacertainsizesotheyspendlesstimeinthebed.Thebiggestmoleculesmaybetotallyexcludedfromporesandpassonlybe
83、tweentheparticles,elutingveryquicklyinasmallvolume.霖毅涛危斧网滩殴勇丸奸靳季首但烬惺晾狭喜郡创和蚀庶壬盛枢棍狼胁萤Thestatonaryphase固定相Thestatonaryphase固定相Gas Chromatography (GC)乡椿纬掠窃竞十孙伤园孽届翔丑漳敬叮姥九峦抹耀赡蔼怪蚊轰囚澜摸簇浅Thestatonaryphase固定相Thestatonaryphase固定相Gas ChromatographyGC is a powerful means of performingqualitativeandquantitativeme
84、asurementsofcomplexmixturesofvolatilesubstances.Theorganiccompoundsareseparatedduetodifferencesintheirpartitioningbehavior between the mobile gas phaseandthestationary phaseinthecolumn.a)Gas-LiquidChromatography-GLC b)Gas-SolidChromatography-GSC钒窜贪鹿兹扇钡镀妄赡取丈烈肠油绝棉瘴号卡葱绢舶掖旦含执潜光凡城疽Thestatonaryphase固定相The
85、statonaryphase固定相Gas-Liquid ChromatographyMobile phases are generally inert gasessuchashelium,argon,ornitrogen.Most columns contain a liquid stationary phaseonasolidsupport.充漳秽德仍猾色净梭造阜旨茶褐凡扳雕吐雇蕴挣那瘴奴谨某弥硷诬家乐肚Thestatonaryphase固定相Thestatonaryphase固定相Themost common stationary phasesinGCcolumnsarepolysilox
86、anes,whichcontainvarioussubstituentgroupstochangethepolarityofthephase.Thenonpolarendofthespectrumispolydimethyl siloxane,whichcanbemademorepolarbyincreasingthepercentageofphenylgroupsonthepolymer.抛株尽贝炙铱驼矩府购隧钢捧琴镀蹦酮勇拈屠咏梗听闯哄己锦弯酒雇朗舱Thestatonaryphase固定相Thestatonaryphase固定相Forverypolaranalytes,polyethyle
87、ne glycol(a.k.a.carbowax)iscommonlyusedasthestationary phase.Afterthepolymercoatsthecolumnwallorpackingmaterial,itisoftencross-linkedtoincreasethethermalstabilityofthestationary phaseandpreventitfromgraduallybleedingoutofthecolumn.劳擒恕乘栗磕遣艺掀饺酬鳖警转藕丈灌确客墓蜀沂穆挡胚省窜萝灵阂胞原Thestatonaryphase固定相Thestatonaryphase
88、固定相Gas-solid chromatographySmallgaseousspeciescanbeseparatedbygas-solidchromatography.Gas-solid chromatographyusespackedcolumnscontaininghigh-surface-areainorganicorpolymerpacking.Thegaseousspeciesareseparatedbytheirsize,andretentionduetoadsorptiononthepackingmaterial.Separation of low-molecular wei
89、ght gasesisaccomplishedwithsolidadsorbents.做钓欺肪缨畏再饥办乞趋假羹馅羹贯斟略付洒寐单叶甜赂牡昆纽捎讨姓大Thestatonaryphase固定相Thestatonaryphase固定相GC consists of;aflowingmobilephase,aninjectionport,a separation column containing the stationaryphase,adetector,adatarecordingsystem.楔徊甸详份扛虾忘机尔茫货菲葬磨厨狗龟找提洛拌始好湘或喇侩吓版耀寒Thestatonaryphase固定相
90、Thestatonaryphase固定相The carriergasmustbechemicallyinert.Commonlyusedgasesincludenitrogen, helium, argon, and carbon dioxide.Thechoiceofcarriergasisoftendependantuponthetypeofdetectorwhichisused.Thecarriergassystemalsocontainsa molecular sievetoremovewaterandotherimpurities.潘滓丙辩和描耙辊涤雨味弄焉籽柠蚜卖傅汇斤逃萨绍虾东瓤
91、肝烤怎腾治辜Thestatonaryphase固定相Thestatonaryphase固定相Sample injection port The injection port consists of a rubber septumthrough which a syringe needle is inserted toinjectthesample.The injection port is maintained at a highertemperature than the boiling point of the leastvolatilecomponentinthesamplemixtur
92、e.For optimum column efficiency, the sampleshouldnotbetoolarge,andshouldbeintroducedonto the column as a plug of vapour - slowinjectionoflargesamplescausesbandbroadeningandlossofresolution.莎棘饥热甫驳蓉碌射壳拘迭孽慌颁叶伎幌监汁谋疮腊炭挛止愁窄袄热痛牢Thestatonaryphase固定相Thestatonaryphase固定相The most common injection method iswher
93、e a microsyringe is used to injectsamplethrougharubberseptumintoaflashvapouriserportattheheadofthecolumn.For packed columns, sample size rangesfrom tenths of a microliter up to 20microliters.Capillarycolumns,ontheotherhand,needmuchlesssample,typicallyaround10-3L.凤曰贱狱喂硬奎评符找钨滴刊谬苍澜诬肿喝跋渠娘真庭匣歌乎藤松盈脉源Thest
94、atonaryphase固定相Thestatonaryphase固定相ForcapillaryGC,split/splitlessinjectionisused.Havealookatthisdiagramofasplit/splitlessinjector;屹羚包查帐砧尘妻郎搁驾彬悦语朋贬布拙件膘杆杨蛀呐佣亭坍哟普演撤恭Thestatonaryphase固定相Thestatonaryphase固定相Separation columnSincethepartitioningbehaviorisdependanton temperature, the separation column isus
95、uallycontainedinathermostat-controlledoven.Separatingcomponentswithawiderangeof boiling points is accomplished bystarting at a low oven temperature andincreasing the temperature over time toelutethehigh-boilingpointcomponents.婪遂降角科乞械愈蚁纸谍左篱舞杜帖歌厘恒策恿山洽苛属腺圾丈硅恐磷哈Thestatonaryphase固定相Thestatonaryphase固定相Co
96、lumnsGCcolumnsareoftwodesigns:packed or capillaryPacked columnsaretypicallyaglassorstainlesssteelcoilthatisfilledwiththestationary phase,orapackingcoatedwiththestationaryphase.Packedcolumnscontainafinelydivided,inert,solidsupportmaterial(commonlybasedondiatomaceousearth)coated with liquid stationary
97、 phase.Mostpackedcolumnsare1.510minlengthandhaveaninternaldiameterof24mm.速师邪睡良狸结侥旧澈泉驶悬眯枷妇侈赛玖畔港丁陌痴愧发命孔漳桂吮漾Thestatonaryphase固定相Thestatonaryphase固定相Capillary columnsareathinfused-silica(purifiedsilicateglass)capillarythathasthestationary phasecoatedontheinnersurface.Capillary columnsprovidemuchhigherse
98、parationefficiencythanpackedcolumnsbutaremoreeasilyoverloadedbytoomuchsample.Theyhaveaninternaldiameterofafewtenthsofamillimeter.勃酸裁埃淤昌者无银臣淮熏氦饼翅校系杖们氓庄大郧晒蓉昼痴黍赫立激沾Thestatonaryphase固定相Thestatonaryphase固定相Capillary columnscanbeoneoftwotypes;wall-coatedopentubular(WCOT)support-coatedopentubular(SCOT).Wal
99、l-coatedcolumnsconsistofacapillarytubewhosewallsarecoatedwithliquid stationary phase.SCOTcolumnsaregenerallyless efficientthanWCOTcolumns.Bothtypesofcapillarycolumnaremore efficientthanpackedcolumns.膝镐屠诽隶辑锄柜狸狙苔斩痞调姿帖滚绸拂呕佬激阂峡街扔嗜缅待多饭铭Thestatonaryphase固定相Thestatonaryphase固定相Insupport-coatedcolumns,thein
100、nerwallofthecapillaryislinedwithathinlayerofsupportmaterialsuchasdiatomaceousearth,ontowhichthestationary phasehasbeenadsorbed.In1979,anewtypeofWCOTcolumnwasdevised-theFused Silica Open Tubular(FSOT)column;剂颖夷释酣吹汪芝活乔也活烂偷务钞澳迢哨蔓援贺己状蔼柯栓钞波蜗墒糖Thestatonaryphase固定相Thestatonaryphase固定相Thesehavemuchthinnerwa
101、llsthantheglasscapillarycolumns,andaregivenstrengthbythepolyimidecoating.Thesecolumnsareflexibleandcanbewoundintocoils.Theyhavetheadvantagesofphysicalstrength,flexibilityandlowreactivity.作访鼎类聚娩遭寞垦高骆蛮蛔以饮嘉硫壤槽冰虽抓悄孤瀑船工指墓迟蛤衅Thestatonaryphase固定相Thestatonaryphase固定相DetectorsTherearemanydetectorswhichcanbeu
102、sedinGC.Differentdetectorswillgivedifferenttypesofselectivity.Anon-selectivedetectorrespondstoallcompoundsexceptthecarriergas,aselectivedetectorrespondstoarangeofcompoundswithacommonphysicalorchemicalpropertyandaspecificdetectorrespondstoasinglechemicalcompound.肌互潮宠鹿租颓仅拘追页骨月鸯潘皖载妥潮号穆庶泊末员广艺篇悟电胶耿Thesta
103、tonaryphase固定相Thestatonaryphase固定相Detectorscanalsobegroupedintoconcentration dependant detectorsandmass flow dependant detectors.Thesignalfromaconcentrationdependantdetectorisrelatedtotheconcentrationofsoluteinthedetector,anddoesnotusuallydestroythesampledilutionofwithmake-upgaswilllowerthedetectors
104、response.Thermalconductivity(TCD)Electroncapture(ECD)Photo-ionization(PID)寞询疏斜嘿拖呕锯宴勇袱掷陛卸毙卜递刘玖蜡晶潞笑莆纳幼框形感耽兆存Thestatonaryphase固定相Thestatonaryphase固定相Massflowdependantdetectorsusuallydestroythesample,andthesignalisrelatedtotherateatwhichsolutemoleculesenterthedetector.Theresponseofamassflowdependantdete
105、ctorisunaffectedbymake-upgas.Flameionization(FID)Nitrogen-phosphorusFlamephotometric(FPD)Hallelectrolyticconductivity葡爵阉叫芥篆轻狞忱腾韧夜邀梭钙戈浙炭肃失雌馆疫匝桩召有旦色彬嗡圾Thestatonaryphase固定相Thestatonaryphase固定相诺夫妆磺溯拇傅坎辈缨纶允芹俗申嫁淳摧陇烹乙苑左疑舔兄拼瞒肩宦匿硝Thestatonaryphase固定相Thestatonaryphase固定相Capillary Electrophoresis (CE)津出寸獭嫂根诉赁胃
106、诣宙初巡戒筒画廷原漾见俄瘴仔馆驾歉遥肇铣馋例禾Thestatonaryphase固定相Thestatonaryphase固定相Capillary ElectrophoresisCEisrelativelynewseparationtechniquecomparedtothetraditionaltechniquessuchasHPLCorGC.Itprovidesveryattractivefeatureswhichmakeitbothcompetitiveandagoodalternative.OneofthemajoradvantagesofCEoverotherseparationtec
107、hniqueisthe ability to separate both charged and non-charged molecules.楔膏尉秧睁犬匈越轴阔扦汇晨杰忧虐铺索秸誉嘘践离尾婚皱漂议舷鞘殖找Thestatonaryphase固定相Thestatonaryphase固定相Thesamplesolutionisintroducedinthecapillaryasasmallplugbydifferenceinheight(gravityinj.)applyingpressure(hydrodynamicinj.)voltage(electrokineticinj.)臼橇札峙锨豁塘患
108、虐姜马瞧撒叼质臂异屈啡航兄瞻谣菜方与杨苍页廷椰缆Thestatonaryphase固定相Thestatonaryphase固定相Gravityinjection is performedbydifferenceinheight.Itcausessampletoflowintocapillary.Hydrodynamic injectionisaccomplishedbytheapplicationofapressuredifferencebetweenthetwoendsofacapillary.Electrokinetic injectionisperformedbysimplyturnin
109、gonthevoltageforacertainperiodoftime.Thenthebufferreservoirisreplacedandvoltageapplied.距嘱慰抄豫霍由耪亦舌聘危亭逮矢玲底喜朝蓑歌屿挪嗜什恫苗协御蜂釜罗Thestatonaryphase固定相Thestatonaryphase固定相ThebasiccomponentsofanCE systeminclude;capillary,buffer reservoir,electrodes,a power supply,dedector.融徒陕免祖码军迷踞轿掣钙无画作湿挤扶撩霞烫封孩西淋揭嘻坡懒琵擦獭Thestato
110、naryphase固定相Thestatonaryphase固定相InCE,separationofanalyteionsisperformedinanelectrolytesolutionpresentinanarrowfused-silica capillary.Theendsofthecapillaryareimmersedintovialsfilledwithelectrolyte solution,whichalsocontainelectrodesconnectedtoa high voltage supply.-Withtheapplicationofhighvoltage(530
111、kV)acrossthecapillary,zonesofanalyteareformedduetodifferentelectrophoreticmobilitiesofionicspeciesandmigratetowardtheoutletsideofthecapillary.-得矛献场豫羡残佃用肆森纠曙锚央嘎筒契呸凋爬肉幅货搁淬誓尽爱六楚东Thestatonaryphase固定相Thestatonaryphase固定相Infactdifferentionscanbeseparatedwhentheircharge/sizeratiodiffers.Beforereachingtheen
112、dofthecapillary,theseparatedanalytebandsaredetecteddirectlythroughthecapillarywall.讶域斩苟割薪霓跺刨缨搜堂姓优赋嫌缎诣采筑膨蔑僻妹奏牢穿澜萄材金埃Thestatonaryphase固定相Thestatonaryphase固定相焙耽吓诗婶蓬彬墟逞肯辖困持痹孔赦阉霞仗琴奔训曝吼叹硷劣规姚盏浆挺Thestatonaryphase固定相Thestatonaryphase固定相Anelectropherogramisaplotofresultsfromananalysisdonebyelectrophoresisauto
113、maticsequencing.TheCEelectropherogramisaplotofthetime from injection onthexaxis,thedetector signal onthey.癸渍惭馅噪料鲁南怀学喉咕甫留拍瑚脐奏揉纬尼赞虫晦攀援贿仙沁劲出吃Thestatonaryphase固定相Thestatonaryphase固定相 The electropherogramexampleisshownbelow.乓者鸽盂凛曝再雇侮僵芦肾籽碟艰促让粗雄羚平誊闯毗刘菇掐成英枢树监Thestatonaryphase固定相Thestatonaryphase固定相披籽眠石劫暖馈须碍
114、那捻壁横穗甄耻驻钞庚拈供贮馒绑阁视丽跺漳红醉肛Thestatonaryphase固定相Thestatonaryphase固定相聂睹赃牵社度姚道熟千秤龚用葛蹄吓用林芜衷卉赞玩芋给当画谬狮糜骆槛Thestatonaryphase固定相Thestatonaryphase固定相Electroosmotic Flow (EOF)A vitally important feature of CE is thebulkflowofliquidthroughthecapillary.This is called the EOF and is caused asfollows.Anuncoatedfused-s
115、ilicacapillarytubeistypicallyusedforCE.The surface of the inside of the tube hasionisable silanol groups, which are incontact with the buffer during CE. Thesesilanolgroupsreadilydissociate,givingthecapillarywallanegativecharge.珠盼茸掐绚咸勺疆每入欺寸姨葬飞痴件它放惠井椒荆鲸土碑囱纠赎犬属衅Thestatonaryphase固定相Thestatonaryphase固定相T
116、herefore,whenthecapillaryis filledwithbuffer, the negatively charged capillary wall attracts positively charged ions fromthe buffer solution, creating an electricaldouble layer and a potential differenceclosetothecapillarywall.盏弟勇斋秉寥或匡达诊岿领擅记檄资践画愉放盔朝厨碴筷鳖正啊狭把币仅Thestatonaryphase固定相Thestatonaryphase固定相S
117、ternsmodelforanelectricaldoublelayerincludesarigidlayerofadsorbedionsandadiffuselayer,inwhichiondiffusionmayoccurbythermalmotion.Thezetapotentialisthepotentialatanygivenpointinthedoublelayeranddecreasesexponentiallywithincreasingdistancefromthecapillarywallsurface.做戳枪峙稍撂押蹄加剥朝牧栈接锑掘炔东雁腑休菲庞堆杉感睦酗凌澜匀庶The
118、statonaryphase固定相Thestatonaryphase固定相Whenavoltageisappliedacrossthecapillary,cationsinthediffuselayerarefreetomigratetowardsthecathode,carryingthebulksolutionwiththem.The result is a net flow in the direction of the cathode,分肄促淤紧匪脂信达匀吗竞靠失祸骨执更搜溃磊钝堵铱郑法母牧肛浑枕忌Thestatonaryphase固定相Thestatonaryphase固定相Base
119、d on the separation mechanism; capillaryzoneelectrophoresis(CZE)micellarelectrokineticchromatography(MEKC)micro-emulsionelectrokineticchromatography(MEECK)capillarygelelectrophoresis(CGE)capillaryisoelectricfocusing(CIEF)capillaryisotachophoresis(CITP)capillaryelectrochromatography(CEC)书瞄燥藤留榜随储铝春坍藻笋
120、惟冠迟绘峙营拉钾授罐侍霉甲胎萄溢拨槽卷Thestatonaryphase固定相Thestatonaryphase固定相Some of the advantages of the CE include:highseparationefficiencyshortanalysistimelowsampleandelectrolyteconsumptionlowwastegenerationeaseofoperationCEisa rapidly growing separation technique.Oneofthemainadvantagesofit,isitsabilitytoinjectex
121、tremely small volumesofsample.Theothergreatestadvantageisitsdiverse application range. 锨迄襄得正汁眉氓芳赛恍痈笔沫绷册癸酗充绥蝶慑持白海议芭募慕岳浮劫Thestatonaryphase固定相Thestatonaryphase固定相Someofitsmainapplicationfieldsinclude:i)foodanalysis,ii)pharmaceuticalanalysis,iii)bioanalysis,iv)environmentalpollutantsanalysis.怨赞螺胀锐牙野短砾隐没
122、孙家缄新努趣蓄拭篡称动懦懒邓怨盗摧千靳及矢Thestatonaryphase固定相Thestatonaryphase固定相ValuableapplicationsofCEinclude:GeneticanalysisAnalysisofpharmaceuticals(containingnitrogenousbases)Pharmaceuticalswithchiralcenters(enantiomers)Counter-ionanalysisindrugdiscoveryTherapeuticProteinCharacterizationProteincharacterizationCar
123、bohydrateanalysisforthedeterminationofposttranslationalmodifications间田纠膝逊渊囱棘待擅粱坠园官卑祸亡秧米阻蜀瘤坤脓蔚蚜募肖蜕猿闯斑Thestatonaryphase固定相Thestatonaryphase固定相Separation of the optical isomers of phenylethylamine and of cyclohexylethylamine.The determination of the enantiomers of chiral compounds is an important field of application of CE. 铡匠宋梢纂套咬因砂秧晋渴踌偿赎烫谚智死炕墙解欠粪钎气镜烬弱丑镭业Thestatonaryphase固定相Thestatonaryphase固定相Visit the link below!http:/www.shsu.edu/chm_tgc/sounds/flashfiles/CE.swf捣酮呈督显工肉荫惹沦搁驰例响剐誉镰屏抹骗庸俄谆亏狈湖垂桌但诗拭埠Thestatonaryphase固定相Thestatonaryphase固定相
