分析化学中常用的分离富集方法

1、分析化学中常用分离和富集方法分析化学中常用分离和富集方法Separation and Preconcentration Methods in Analytical ChemistryintroductionSeparation sciencephysicschemistryLife scienceMaterial scienceChemical Engineering Medicine & pharmaceuticsJournals of separation science(analytical chemistry)Lab on a ChipJ CHROMATOGR A ELECTR

2、OPHORESIS J CHROMATOGR B J SEP SCI CHROMATOGRAPHIA J CHROMATOGRAPHY SCI ENCEJ LIQ CHROMATOGR R TJPC-J PLANAR CHROMAT ACTA CHROMATOGR LC GC N AM Performance %100%原始含量分离后测量值）回收率（ Chemical separationsGas separation （气态分离）（气态分离）Precipitation (沉淀沉淀)Extraction （萃取）（萃取）Ion exchange（离子交换）（离子交换）Chromatograph

3、y （色谱）（色谱）Elelctrophoresis （电泳）（电泳）Foam fractionation （气浮）（气浮）Membrane Separation （膜分离）（膜分离）Methods to be covered1.气态分离法气态分离法挥发与升华挥发与升华 （evaporation and sublimation ） 挥发：固体或液体全部或部分转化为气体的过程挥发：固体或液体全部或部分转化为气体的过程 氢气还原砷酸盐转为氢气还原砷酸盐转为AsH3飞硅（飞硅（SiF4）飞铬飞铬(CrO2Cl2)升华升华: 固体物质不经过液态就变成气态的过程固体物质不经过液态就变成气态的过程Dist

4、illation （蒸馏）（蒸馏） a 常压蒸馏常压蒸馏 b 水蒸气蒸馏水蒸气蒸馏当混合物中各组分蒸气压总和等于外界大气压时，这时的温度即为当混合物中各组分蒸气压总和等于外界大气压时，这时的温度即为它们的沸点。此沸点比各组分的沸点都低。它们的沸点。此沸点比各组分的沸点都低。 如果一溶液的组成在它的沸点分解，必须减压蒸馏它或水蒸汽蒸馏如果一溶液的组成在它的沸点分解，必须减压蒸馏它或水蒸汽蒸馏它，水蒸气蒸馏的那些化合物须不与水混溶它，水蒸气蒸馏的那些化合物须不与水混溶c 减压和真空蒸馏减压和真空蒸馏 在大气压以下的蒸馏称为减压和真空蒸馏在大气压以下的蒸馏称为减压和真空蒸馏例如无水乙醇的制备，水和乙

5、醇形成共沸物例如无水乙醇的制备，水和乙醇形成共沸物(95乙醇乙醇)，b.p.=78.15加入苯形成另一共沸物加入苯形成另一共沸物(苯苯74，乙醇，乙醇18.5,水水7.5%) b.p.=65在在65蒸馏蒸馏, 除去水除去水, 在在68苯和乙醇形成共沸物苯和乙醇形成共沸物(苯苯67.6,乙醇乙醇32.4)在在68蒸馏直到温度升高，在蒸馏直到温度升高，在78.5能获得纯乙醇。能获得纯乙醇。 d 共沸蒸馏共沸蒸馏e 萃取蒸馏萃取蒸馏(extractive distillation)例由氢化苯例由氢化苯(80.1)生成环己烷生成环己烷(80.8)时时,一般的蒸馏不能分离，加入一般的蒸馏不能分离，加入苯

6、胺苯胺(184)与苯形成络合物，在比苯高的温度沸腾，从而分离环己烷与苯形成络合物，在比苯高的温度沸腾，从而分离环己烷 2. 沉淀沉淀 （Precipitation separationgroupreagentHClH2SO4NH4Cl - NH3NaOHsoluble*ionAg Hg (I) (Pb)Ca Sr Ba PbFe (III) Mn (II) Al Hg (II) Cr (III)CuMgCdCoNiNaKZnNH4+有机沉淀剂有机沉淀剂 草酸草酸: 沉淀沉淀Ca, Sr, Ba, RE, Th 铜铁试剂铜铁试剂(N-亚硝基苯基羟铵亚硝基苯基羟铵): 强酸中沉淀强酸中沉淀Cu,F

7、e,Zr,Ti,Ce.Th,V,Nb,Ta等，微酸中沉等，微酸中沉淀淀Al,Zn,Co,Mn,Be,Th,Ga,In,Tl等。主要用于等。主要用于1:9的硫的硫酸介质中沉淀酸介质中沉淀Fe(III),Ti(IV),V(V)等与等与Al,Cr,Co,Ni分离分离 铜试剂铜试剂 (二乙胺基二硫代甲酸钠，二乙胺基二硫代甲酸钠，DDTC) 沉淀沉淀Cu,Cd,Ag,Co,Ni,Hg.Pb.Bi,Zn等重金属离子，与等重金属离子，与稀土、碱土金属离子及铝等分开稀土、碱土金属离子及铝等分开痕量组分的富集和共沉淀分离痕量组分的富集和共沉淀分离 无机共沉淀剂进行共沉淀无机共沉淀剂进行共沉淀 有机共沉淀剂进行共

8、沉淀有机共沉淀剂进行共沉淀 l 利用表面吸附进行痕量组分的共沉淀富集利用表面吸附进行痕量组分的共沉淀富集, 选择性选择性不高。共沉淀剂为不高。共沉淀剂为Fe(OH)3, Al(OH)3等胶状沉淀等胶状沉淀, 微微溶性的硫化物，如溶性的硫化物，如Al(OH)3作载体共沉淀作载体共沉淀Fe3 +,TiO2+; HgS共沉淀共沉淀Pb2+l 利用生成混晶进行共沉淀，选择性较好，如硫酸铅利用生成混晶进行共沉淀，选择性较好，如硫酸铅-硫酸鋇，磷酸铵镁硫酸鋇，磷酸铵镁-砷酸铵镁等砷酸铵镁等l 利用胶体的凝聚作用进行共沉淀利用胶体的凝聚作用进行共沉淀, 如动物胶、丹宁如动物胶、丹宁l 离子缔合共沉淀，如甲基

9、紫与离子缔合共沉淀，如甲基紫与InI4-。l 利用利用“固体萃取剂固体萃取剂”进行共沉淀，例进行共沉淀，例 1-萘酚的乙醇溶萘酚的乙醇溶液中，液中，1-萘酚沉淀，并将萘酚沉淀，并将U(VI)与与1-亚硝基亚硝基-2-萘酚的螯萘酚的螯合物共沉淀下来。合物共沉淀下来。 在含有在含有1.010-2mol/L Al3+和和 1.010-2mol/L Zn2+的溶液中，的溶液中，加入氨性缓冲溶液，使加入氨性缓冲溶液，使NH3为为0.2 mol/L和使和使NH4+为为1.0 mol/L，问此时两离子能否定量分开？问此时两离子能否定量分开？解：解： 要两离子分离完全，必须是一离子沉淀完全，另一离子不要两离子

10、分离完全，必须是一离子沉淀完全，另一离子不沉淀。从题意看出，沉淀。从题意看出，Al(OH)3溶解度小，通过计算溶解度小，通过计算,看看Al(OH)3沉淀后，残留在溶液中的沉淀后，残留在溶液中的Al3+浓度是否小于其起始浓度的浓度是否小于其起始浓度的1/1000，且，且Mg2+不沉淀。先计算氨性溶液中的不沉淀。先计算氨性溶液中的OH- 。根据反应根据反应NH3H2O = NH4 + OH-OH- = KbNH3cNH3/cNH4+ =1.810-50.20/1.0 = 3.610-6（mol/L） Mg2+OH-2=1.010-2(3.610-6)21.310-13 KspAl(OH)3离子浓度

11、积计算看出，离子浓度积计算看出，Mg2+不沉淀，不沉淀，Al3+沉淀。沉淀。Al3+沉淀是否沉淀是否完全要看残留在溶液中的完全要看残留在溶液中的Al3+是否小于起始浓度的是否小于起始浓度的1/1000。即即1.010-210-31.010-5Al3+= KspAl(OH)3/OH-3 = 1.310-33/(3.610-6)3 2.810-17（mol/L）远小于远小于1.010-5，所以认为，所以认为Al3+沉淀完全，两离子得以完全分沉淀完全，两离子得以完全分离。离。 A significant method based on relative solubility of an analyt

12、e in two immiscible liquidsUsed to Remove interference Concentrate species prior analysis Produce measurable form of a speciesBasic theory is applicable to chromatography3. Extraction (萃取分离法萃取分离法)3.1 Solvent ExtractionPrinciple of the extraction separation例，例，8-羟基喹啉羟基喹啉-CHCl3对对Al 3+ 的萃取的萃取extraction

13、 of Al 3+ with 8-hydroxyquinoline -CHCl3Al(H2O)6 3+ 3 NOHAlNO+ 3 H+ 6 H2O 3hydrophilichydrophobicSoluble in CHCl38-hydroxyquinolineCHCl3Extraction reagentsolventpartition coefficient and distribution ratiowDKHAHAoPartition lawKDpartition coefficient (分配系数分配系数)Conditional partition coefficientwCCDHA,

14、oHA,wHAwoHA,oHAHAwHAoHADK,oHA,wHA,The percent extractedwoommmwwooooVcVcVcowwowoVVccccRDDowVVR Phase ratio1DDEDE %1001.00.01100500D1101001000E %50919999.9Improve EDecrease RIncrease extraction timesEmultiple extractionwowoVmVmmCCD/ )(110 wowVDVVmm01After n times extractionnwownVDVVmm)(000mmmEnpercent

15、 extractedVo =100 mL, m0 = 0.20 g, D = 85Extraction types and conditionsCH3C N OHC N OHCH3+Ni2+CH3C NOCCH3NOHCH3CNOC CH3NOHNi+ 2H+ 2Extraction with CHCl3Dithizone 双硫腙双硫腙8-hydroquinoline and cupferronNOHNO-NO8-hydroquinoline8-羟基喹啉羟基喹啉Extraction equilibriumExtraction M with HLnHLDnHLanMLDexKKKKn)()()(

16、)(nMLDKnnHLaK)(nHLDK)(exKM(w) + n (HL)(o) = MLn (o) + n H+(w)nnnexKowwoHLMHMLnThe effects of acidity on chelate extractionnownwoexKHLMHMLn nonwDHLHnwnoexKDHHLnwnoexwonKMMLDHLH nwnonoHLwMexKHLH)()(Dithizone 100500pHE%0246810今有今有100 ml 1.010-5mol/L的金属离子（的金属离子（M2+）水溶液，在）水溶液，在pH=5.00时有时有33的以的以ML2形式萃入到形式

17、萃入到20 ml 1.010-3mol/L的的HL有机相中。问有机相中。问100 ml M2+、在、在pH6.00时，用时，用50ml 5.010-4mol/L HL的有机相萃取，其萃取率是多少？的有机相萃取，其萃取率是多少？ 先求分配比（先求分配比（D） 在在pH5.00时，时，E=33%,V水水100mL，V有有20mL，根据公式，根据公式 根据萃取平衡常数根据萃取平衡常数(K)公式计算公式计算pH=5.00时萃取平衡常数时萃取平衡常数 计算计算pH6.00时的萃取率时的萃取率将将H+=10-6, K=2.510-4，Vw=100 ,Vo=50 ,HLo=5.010-4mol/L带入得：带

18、入得： ion association extraction （离子缔合物萃取）（离子缔合物萃取）Extracted by (CH3CH2)2ONSN(CH3)2(CH3)2N +BF4-Techniques of extraction间歇萃取法间歇萃取法连续萃取法连续萃取法Continuous liquid-liquid extractionHigher-density solvent extractionLower-density solvent extractionsoxhlet extraction (索氏萃取索氏萃取)3.2 Solid phase extraction (固相萃取固

19、相萃取)3.3 Solid phase microextraction (固相微萃取固相微萃取)3.4 Supercritical fluid extraction (超临界流体萃取超临界流体萃取)Nature 2000, 405, 129-130原理：原理： 超临界流体萃取超临界流体萃取:气气-固萃取固萃取 萃取剂萃取剂: 超临界条件下的气体（液体）超临界条件下的气体（液体） 粘度低，接近零的表面张力，比很多液体容易渗透固粘度低，接近零的表面张力，比很多液体容易渗透固体颗粒，易于除去。体颗粒，易于除去。 3.5 Single drop microextraction (单滴微萃取单滴微萃取)

20、离子交换树脂4. 离子交换分离离子交换分离 Ion exchange离子交换分离法是离子交换剂与溶液中的离子交换分离法是离子交换剂与溶液中的离子发生交换反应而使离子分离的方法离子发生交换反应而使离子分离的方法离子交换树脂的结构离子交换树脂的结构带有活性基团的网状高分子聚合物带有活性基团的网状高分子聚合物骨架骨架活性基团活性基团酚醛树脂酚醛树脂聚乙烯树脂聚乙烯树脂OH + CH2OCH=CH2+CH=CH2CH=CH2交联剂交联剂酸性基团酸性基团碱性基团碱性基团SO3HCOOHN+R3OH- NR2Ion exchange resins特殊基团特殊基团聚苯乙烯磺酸型阳离子交换树脂聚苯乙烯磺酸型阳

21、离子交换树脂CH=C H2+CH=C H2CH=C H2H2SO4CHCH2CHCHCH2CH2CH2CHCHCH2CHCHCH2CH2CH2CHCHCH2CHCHCH2CH2CH2CHCHCH2CHCHCH2CH2CH2CHSO3HSO3HSO3HSO3H聚合磺化交联剂交联剂交联作用交联作用活性基活性基团团R-SO3H + M+ = R-SO3M + H+离子交换树脂的分类离子交换树脂的分类依据活性基团分类依据活性基团分类阳离子交换树脂阳离子交换树脂阴离子交换树脂阴离子交换树脂螯合树脂螯合树脂特殊交换树脂特殊交换树脂强酸型强酸型弱酸型弱酸型交换基为酸性，交换基为酸性，H+与阳离子交换与阳离子

22、交换SO3HCOOHOHpH 2pH 6使用使用 pH 范围范围pH 10交换基为碱性，交换基为碱性，阴离子发生交换阴离子发生交换强碱型强碱型弱碱型弱碱型N+(CH3)3OH-N+H3 OH-N+H2R OH-N+HR2 OH-pH 12pH pKapOHpKbMolecular imprinting 分子印迹分子印迹Exchange reaction 阳离子交换树脂阳离子交换树脂阴离子交换树脂阴离子交换树脂R-SO3H + M+ R-SO3M + H+RN+(CH3)3OH- + X- RN+(CH3)3X- + OH-R-NH2 + H2O R-N+H3 OH- 水合作用水合作用RN+H3

23、OH- + X- RN+H3X- + OH-螯合交换树脂螯合交换树脂R-L + M (R-L)nMPerformance of ion exchange resin交联度交联度交换容量交换容量表征骨架性能的参数表征骨架性能的参数表征活性基团的性能参数表征活性基团的性能参数是指交联剂在反应物中所占的质量分数是指交联剂在反应物中所占的质量分数氨基酸的分离，交联度氨基酸的分离，交联度8 %， 多肽的分离多肽的分离 2 4 %每克干树脂所能交换的物质的量（每克干树脂所能交换的物质的量（mmol)。决定于网状结构中活性基团的数目。决定于网状结构中活性基团的数目。交换容量由实验测得交换容量由实验测得交换容

24、量的测定交换容量的测定IVV0流出曲线流出曲线始始漏漏量量交换过程与总交换量交换过程与总交换量 exchange capacity总交换容量总交换容量 是指柱上树脂所能交换的总量。是指柱上树脂所能交换的总量。离子交换亲合力离子交换亲合力 affinityR-A+ + B+ R-B+ + A+ BAABKrr)()(AKBKKDDwDBKBB)o（亲和力亲和力KK 强酸性阳离子交换树脂强酸性阳离子交换树脂 Li+ H+ Na+ NH4+ K+ Rb+ Cs+ Ag+ Tl+ U(VI)Mg2+Zn2+Co2+Cu2+Cd2+Ni2+Ca2+Sr2+Pb2+Ba2+ Na+Ca2+Al3+Th4+

25、 对于弱酸性阳离子交换树脂，对于弱酸性阳离子交换树脂，H的亲合力大于阳离子的亲合力大于阳离子 a. 阳离子交换树脂阳离子交换树脂b. 阴离子交换树脂阴离子交换树脂强碱型阴离子交换树脂强碱型阴离子交换树脂F-OH-CH3COO-HCOO-Cl-NO2-CN-Br-C2O42- NO3- HSO4- I- CrO42- SO42- Cit.强碱型阴离子交换树脂强碱型阴离子交换树脂F-Cl-BrI-CH3COO-MoO42-PO43-AsO43-NO3-Tart.CrO42- SO42- OH-2.0 g H+树脂，与含有树脂，与含有0.0010 mol/L Ca2+的的100 ml 0.10 mo

26、l/LHCl一起振荡，交换达到平衡时，一起振荡，交换达到平衡时，Ca2+残留在溶液中的残留在溶液中的百分率？。已知交换平衡常数百分率？。已知交换平衡常数K3.2，树脂交换容量为，树脂交换容量为5 mmol/g。树脂上浓度用。树脂上浓度用mmol/L表示，溶液浓度用表示，溶液浓度用mmol/mL表示。表示。 解：解：1mol Ca2+可以交换可以交换2mol H+，假如大部分，假如大部分Ca2+进入树进入树脂 ，脂 ， C a2 +和和 H+在 树 脂 上 和 溶 液 中 的 浓 度 分 别 为在 树 脂 上 和 溶 液 中 的 浓 度 分 别 为Ca2+r,H+r,Ca2+,H+。 树脂上树脂

27、上H+r为：为： H+r=(5.02.00.00101002)/2.0 = 4.9(mmol) 溶液中溶液中H+H+=0.10+20.00100.102（mmol/ml）已知交换平衡常数已知交换平衡常数K3.2，即，即 已知交换平衡常数已知交换平衡常数K3.2，即，即 Ca2+在树脂和溶液两相间的分配比在树脂和溶液两相间的分配比DCa为为溶液体积为溶液体积为100ml，树脂质量为，树脂质量为2.0g，留在溶液中的，留在溶液中的Ca2+百分率为百分率为 强酸型阳离子交换树脂分离示例强酸型阳离子交换树脂分离示例Cl-, K+, Na+, Ag+ 的分离的分离K：亲和力：亲和力 H+ Na+ K+

28、Ag+H2O交换交换 exchange游离状态游离状态CtNa+K+Ag+Application Purification of WaterH+OH-Cation exchangeR-SO3H + M+ R-SO3M + H+RN+H3OH- + X- RN+H3X- + OH-H+ + OH- H2O de-ionized wateranion exchangeConcentration of the trace elementsCl-RN+(CH3)3OH-5. Chromatography （色谱分离）（色谱分离）洗脱洗脱nChromatography has application i

29、n every branch of the chemical and biological sciencesnAt least 20 Nobel prizes were awarded for their work in which chromatography played a vital role这是一个区域，这是一个区域，也就是同一种物也就是同一种物质是质是不能够在一瞬间不能够在一瞬间同时析出来同时析出来DefinitionChromatographynA process in which a chemical mixture carried by a liquid or gas is

30、separated into components as a result of differential distribution of the solutes as they flow around or over a stationary liquid or solid phasenEncompasses a diverse and important group of methods that permit scientist to separate closely related components in a mixture通常是同一种溶剂Types of chromatogarp

31、hy (based on shape of the support)ChromatographyPlanarColumnPaperThin Layer(TLC)GAS (GC)Liquid (LC)后一种方法用的更多一些后一种方法用的更多一些Types (based on mobile phase)nGas chromatography (GC)nGas liquidnGas solidnHigh performance liquid chromatography (HPLC)nPartition （分配）nAdsorption （吸附）nIon-exchange （离子交换）nSize-ex

32、clusion （尺寸排阻）nAffinity （亲和）nSupercritical fluid chromatographyTypes (based Separation Mechanism ) Adsorption chromatography Partition chromatography Ion-exchange chromatography Exclusion chromatography (Gel-permeation chromatography) Affinity chromatographyExample of separation mechanisms used in c

33、hromatographyIon-exchange chromatographySeparation is based on exchange of ions between surface and eluents.Partition chromatographySeparation is based on solute partitioning between two liquid phases.Adsorption chromatographySeparation is due to a series of adsorption/ desorption steps.Size-exclusi

34、on chromatographySeparation is based on molecular size.+-专门的一块知识Principle of affinity chromatographyThe analyte (enzyme, antibody, antigen, tissue receptor, etc.) binds to the support-bound ligand. It subsequently is eluted with a general eluent (such as a chaotropic agent), pH change, or biospecifi

35、c eluent (such as an inhibitor or substrate).SupportEluting agentLigandSpacer armFrontSolventFrontFrontElutingsolventSmall molecules retarded (entrapped in beads)Large molecules eluted (bypassed the beads)Gel particleLarge moleculeSmall moleculeSchematic representation of gel-filtration column chrom

36、atpgraphy.Parts of ColumnnColumn（柱）（柱）nsupportnstationary phasenmobile phaseThin-Layer Chromatography（薄层色谱）（薄层色谱）Glass lidTankSolventGlass plate with thin layer of silica gelPoint of sample applicationMovement of solventSolvent frontPoint to which sample migratedThe solvent moves up the thin layer o

37、f absorbent by capillary action. In practice the TLC plate usually is developed as the solvent is allowed to move in an ascending direction.TLC Thin-layer chromatographyCCD摄像摄像paper chromatography （纸色谱）（纸色谱）retardation factor (比移值) Rfl()fAaRl()fBbRlBbaA Separation techniques based on the movement of

38、 analytes through a conductive medium in response to an applied electrical field. The medium is usually a buffered aqueous solution. In the absence of other factors, cationic species will migrate towards the cathode and anionic species will migrate towards the anode. The rate of migration is based o

39、n the charge to size ratio of each species6. Electrophoresis（电泳）（电泳）The biggest limitation of the classic electrophoresis: Joule Heating（使热运动加快）（使热运动加快）for his research on electrophoresis and adsorption analysis, especially for his discoveries concerning the complex nature of the serum proteinsArne

40、Wilhelm Kaurin Tiselius The Nobel Prize in Chemistry 1948Joule Heating: uneven heating in the system by the electric field causes different points in the system to have different temperatures non-uniform mixing of solute and solvent gives rise to peak broadening in electrophoresis decrease band-broa

41、dening caused by Joule heating:1981, capillary with i.d. of 75 m was used by Jorgenson and Lukas for separation of amino acid with N of 400,000 /mthe milestone HPCE.Form 1988, commercial equipments, developed rapidly, various models, various detection methods, and various application fields, acceler

42、ate the CE development recently，capillary array electrophoresis (CAE) chip-based electrophoresis (chip-CE)1967, the CE was firstly proposed by Hjerten by using capillaryHuman Genome ProjectGoals: identify all the approximate 30,000 genes in human DNA, determine the sequences of the 3 billion chemica

43、l base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project. Dideoxy SequencingnDideoxy sequencing (also called

44、 chain- termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the diagram contains.nFor example, i

45、n the A reaction tube the ratio of the dATP to didATP is adjusted so that each tube will have a collection of DNA fragments with a didATP incorporated for each adenine position on the template DNA fragments. The fragments of varying length are then separated by electrophoresis and the positions of t

46、he nucleotides analyzed to determine sequence. The fragments are separated on the basis of size, with the shorter fragments moving faster and appearing at the bottom of the gel. Sequence is read from bottom to top.DNA的复制需要：DNA聚合酶，单链DNA模板，带有3-OH末端的单链寡核苷酸引物，4种dNTP（dATP、dGTP、dTTP和dCTP）。聚合酶用模板作指导，不断地将dN

47、TP加到引物的3-OH末端，使引物延伸，合成出新的互补DNA链。如果加入一种特殊核苷酸，双脱氧核苷三磷酸（ddNTP），因它在脱氧核糖的3位置缺少一个羟基，故不能同后续的dNTP形成磷酸二酯键。如，存在ddCTP、dCTP和三种其他的dNTP（其中一种为-32P标记）的情况下，将引物、模板和DNA聚合酶一起保温，即可形成一种全部具有相同的5-引物端和以ddC残基为3端结尾的一系列长短不一片段的混合物。经变性聚丙烯酰胺凝胶电泳分离制得的放射性自显影区带图谱将为新合成的不同长度的DNA链中C的分布提供准确信息，从而将全部C的位置确定下来。类似的方法，在ddATP、ddGTP和ddTTP存在的条件下

48、，可同时制得分别以ddA、ddG和ddT残基为3端结尾的三组长短不一的片段。将制得的四组混合物平行地点加在变性聚丙烯酰胺凝胶电泳板上进行电泳，每组制品中的各个组分将按其链长的不同得到分离，制得相应的放射性自显影图谱。从所得图谱即可直接读得DNA的碱基序列. 使用的是荧光标记的，每一种上面只显出一种光Brown. Genomes 2荧光属于分子能级Capillary ElectrophoresisnA method was then developed based on this where fluorescent dideoxynucleotides were used in a single

49、 sequencing reaction. The fragments are then separated in a single lane of a sequencing gel and identified by the fluorescent signature. nThe limitations of gel electrophoresis soon became apparent. Gels take a long time to run and have a limited reproducibility. An automated method was much more de

50、sirable, but the automation of gels requires complex robotic handling. nThe use of capillaries allows much higher electrical fields to be used making the separations faster. Flexible capillaries are also easily incorporated into an automated instrument making sequencing cheap, fast and efficient.nHo

51、wever a single capillary is still a bottleneck in the sequencing process. A gel is easily capable of running up to 96 samples simultaneously. To overcome this a instrument fitted with an array of capillaries was developed. In the first instruments the detector moved across the array, but the time la

52、g means some information can be missed. Now all the capillaries are simultaneously monitored using an array of photodiodes.Dovichi, Karger: were appraised as “ unsung hero” in Science magazine “分析科学家拯救了人类分析科学家拯救了人类HGP计划计划”每一种荧光值只特性激发一种荧光物质New generation sequence technique: nanopore（纳米通道测序）（纳米通道测序）ba

53、sic principle of CEApparatus:High-voltage DC power source（HV）: 0 -30 kVelectrode: Pt wirecapillary: quartz (75，50，25，10，5，2，1 m )detector: UV-VIS, ECD, LIF, CL, MS, NMR Characteristics of CE: high performance（N: 105106） high speed（ 20 min，chip-CE: s or ms） low consumption （nL-pL） high sensitivity，si

54、ngle molecules detection with LIF low cost, wide applicationElectrophoretic MobilitynThe movement of ions solely due to the electric field, potential differencenCations migrate toward cathodenAnions migrate toward anodenNeutral molecules do not favor eithervep = epEnep = q/(6r) is buffer viscositynr

55、 is solute radiusnProperties that effect mobility1.Voltage applied2.Size and charge of the solute3.Viscosity of the buffer- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + +Electro

56、osmotic FlownAs the buffer sweeps toward the anode due to the electric field, osmotic flow dictates the direction and magnitude of solute ion flow within the buffernAll ions are then swept toward the anode.nNegative ions will lead the neutral ions toward the anodenPositive ions will trail the neutra

57、l ions as the cathode pulls them整体还是电中性的值得注意的是当两片电极设计成正电荷时，值得注意的是整个电渗就是负电的当两片电极上的电荷减少那么电渗会减小而电泳则可能会和电渗一个数量级上。设计出发点：原来在一边电泳是不太合适的？Electroosmotic Mobilitynveof = eofEneof = / (4 )n = buffer dielectric constantn = zeta potentialnZeta PotentialnThe change in potential across a double layernProportional

58、to the charge on the capillary walls and to the thickness of the double layer.nBoth pH and ion strength affect the mobilityHPCE (flat flow)HPLC (Laminar flow)Total Mobilitynvtot = vep + veofnMigration timesnvtot = l/tnl = distance between injection and detectionnt = migration time to travel distance

59、 lnt = lL/(ep + eof)VnL = length of capillarynV = voltageThe eof is, in general 6-7 folds bigger than ep, so all the species migrate from anode to cathode according the sequence from cations, neutral molecules and anions.Separation efficiency and resolutionN = 5.54 (tm/ w1/2)2N = l 2/ 2 2 = 2Dtm (on

60、ly lognitudinal diffusion is account for the broarding of the peak.)tm = l / ep E = lL/ ep V V l2DL(vef + veo ) N =Means to improve efficiency:（1）high electric field（2）improve EOF（3）small D（biomacromolecules such as DNA, protein etc.)Microfluidic chip（微流控芯片）（微流控芯片） TAS(Micro Total Analysis System ):微全分析