高效液相用法High performance liquid chromatography.docx

高效液相用法High performance liquid chromatography.docx

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高效液相用法Highperformanceliquidchromatography

高效液相用法（Highperformanceliquidchromatography）

HPLCmethodisbasedontheclassicalchromatography,gaschromatographyreferencetheoryintechnology,themobilephasechangedtohighpressure（maximumconveyingpressureupto4.9107Pa）;thecolumnisaspecialmethodforsmallsizefillerfilledup,sothatthecolumnefficiencyismuchhigherthantheclassicalliquidchromatography（numberperMitaboardtensofthousandsorhundredsofthousands）;atthesametimecolumnisconnectedwiththereardetectorwithhighsensitivity,continuousdetectionofeffluent.

Characteristic

1.highpressure:

liquidchromatographyusingliquidasmobilephase（calledcarrierfluid）,liquidflowingthroughthecolumn,theresistanceisgreater,inordertoquicklypassthroughthecolumn,mustbeappliedtotheliquidpressure.Generallyupto150~350*105Pa.

2.highspeed:

theflowvelocityofthemobilephaseinthecolumnismuchfasterthanthatoftheclassicalchromatography,andgenerallyreaches1~10ml/min.TheanalyticaltimerequiredforHPLCismuchlessthanthatofclassicalliquidchromatography,generallylessthan1h.

3.efficient:

Recently,manynewstationaryphaseshavebeendeveloped,whichgreatlyimprovestheseparationefficiency.

4.highsensitivity:

highsensitivityliquidchromatographyhasbeenwidelyusedinhighsensitivitydetectors,whichfurtherimprovesthesensitivityoftheanalysis.Suchasfluorescencedetector,sensitivityupto10-11g.Inaddition,thesamplesizeissmall,andgenerallyslightlyincreased.

5.toadapttoawiderange:

comparisonofgaschromatographyandhighperformanceliquidchromatography,gaschromatographyhasgoodseparationability,highsensitivity,fastanalysisspeed,convenientoperationandotheradvantages,butlimitedtechnicalconditions,thethermalstabilityofmaterialortoohighboilingpointdifferencematterisdifficulttousegaschromatographymethodofanalysis.Highperformanceliquidchromatography（HPLC）requiresthatthesamplebemadeintoasolutionwithouttheneedforgasification,sothatitisnotsubjecttothevolatilityofthesample.Forhighboilingpoint,poorthermalstability,relativelyhighmolecularweight（morethan400）oforganicmatter（thesesubstancesaccountedforalmost75%ofthetotalorganicmatter~80%）principlecanbeappliedbyhighperformanceliquidchromatographyforseparationandanalysis.Accordingtostatistics,intheknowncompounds,gaschromatographycanaccountforabout20%,whileliquidchromatographycanaccountforabout70to80%.

Highperformanceliquidchromatographystationaryphaseaccordingtotheirnaturecanbedividedintohighhydrophobicgelchromatography,high-performanceliquidchromatography,reversed-phasehighperformanceliquidchromatography,ionexchangehighperformanceliquidchromatography,highperformanceliquidchromatographyandhighaffinityliquidchromatographyandothertypesoffocus.Theprincipleofseparationandanalysisofvariouscompoundsbyhighperformanceliquidchromatographyissimilartotheprincipleofcommonliquidchromatography.ThedifferenceisthatHPLCissensitive,fast,highresolutionandreproducible,andhastobecarriedoutinchromatograph.

Maintypesandseparationprincipleofhighperformanceliquidchromatography

Accordingtotheseparationmechanism,HPLCcanbedividedintothefollowingmaintypes:

1.liquidliquidpartitionchromatography（Liquid-liquid,Partition,Chromatography）andchemicalbondedChromatography（ChemicallyBondedPhase）

Themobilephaseandthestationaryphasearebothliquids.Thereisanobviousinterfacebetweenthemobilephaseandthestationaryphase,whicharemutuallyinsoluble（differentpolarityandavoidingthelossofthestationaryliquid）.Whenthesampleentersthecolumn,thesoluteisdistributedbetweentwophases.Whenbalanced,submittothelower:

Intheformula,theconcentrationofCS-soluteinthestationaryphase;theconcentrationofcm--soluteinthemobilephase;thevolumeoftheVs-stationaryphase;andthevolumeofVm-Mobilephase.LLPCandGPChavesimilarities,thatis,theorderofseparationdependsonK,andtheretentionoflargecomponentsofKislarge,buttherearealsodifferences.InGPC,theflowrelativetoKhaslittleeffect,andtheLLPCflowhasagreaterinfluenceonK.

A.NormalPhaseliquidChromatography:

thepolarityofthemobilephaseislessthanthepolarityofthestationaryliquid.

B.reversedphaseliquidliquidpartitionchromatography（ReversePhaseliquidChromatography）:

thepolarityofmobilephaseisgreaterthanthatofstationaryliquid.

C.

Liquid-liquidpartitionchromatographydisadvantages:

Althoughthemobilephaseandstationaryphasepolarityrequirementsarecompletelydifferent,butfixedliquidinthemobilephaseisstilldissolved;themobilephasebymechanicalshockcolumnforce,willcausethelossofstationaryliquid.Thechemicalbondattheendofthe70sdevelopmentofthelastcenturystationaryphase（seebelow）,thiscanbeovercome.Itisnowwidelyused（70~80%）.

2.liquidsolidchromatography

Themobilephaseisliquid,andthestationaryphaseisadsorbent（suchassilicagel,alumina,etc.）.Thisisbasedontheadsorptionofsubstancestoseparate.Themechanismis:

whenthesampleentersthechromatographiccolumn,thesolutemolecule（X）andsolventmolecules（S）occurrenceofcompetitiveadsorptionontheadsorbentsurfaceactivecenter（notintothesample,alloftheadsorbentadsorptionactivecenterisS）,whichcanbeexpressedasfollows:

Xm+nSa+nSmhistoryXa

Formula:

solutemoleculesinXm--mobilephase;solventmoleculesinSa--stationaryphase;solutemoleculesinXa--stationaryphase;solventmoleculesinSm--mobilephase.

Whentheadsorptioncompetitionreactionreachesequilibrium:

K=[Xa][Sm]/[Xm][Sa]

Formula:

Kistheequilibriumconstantofadsorption.[discussion:

thebiggertheK,thegreatertheretentionvalue.]

3.ionexchangechromatography（Ion-exchange,Chromatography）

IECisanionicexchangerusedasastationaryphase.IECisbasedonreversibleexchangeofionizableionsonionexchangeresinswithsoluteionswiththesamechargeinthemobilephase,whichareseparatedbyexchangeagentswithdifferentaffinities.

Takinganionexchangersasanexample,theirexchangeprocesscanbeexpressedasfollows:

X-（solvent）+（-R4N+Cl-resin）（-R4N+=X-+Cl-resin）（solvent）

Whentheexchangereachesequilibrium:

KX=[-R4N+X-][Cl-]/[-R4N+Cl-][X-]

Partitioncoefficient:

DX=[-R4N+X-]/[X-]=KX[-R4N+Cl-]/[Cl-]

[discussion:

relationshipbetweenDXandretentionvalue]

Anysubstancethatcanionizeinasolventcanusuallybeseparatedbyionexchangechromatography.

4.ionpairchromatography（Ion,Pair,Chromatography）

Isakindofionchromatography（ormore）ioninsteadofsolutemolecules（calledthechargeofionsandcounterions）addedtothemobilephaseorthestationaryphase,whichcombinedwiththeformationofhydrophobicsoluteionionpaircompounds,soastocontroltheretentionbehaviorofsoluteions.Theprinciplecanbeexpressedinthelowerform:

X++Y-=X+Y-aqueousaqueousorganicphase

Type:

X+-phaseflowofaqueousorganicionstobeseparated（butalsocationic）;Y-phase-phaseflowoppositelychargedions（suchashydrogenoxidationoffourtetrabutylammoniumhydroxide,sixteenalkyltrimethylammoniumion;etc.）ontheformationofX+Y---compounds.

Whenthebalanceisreached:

KXY=[X+Y-]/X+][Y-]aqueousaqueousorganicphase

Bydefinition,thepartitioncoefficientis:

DX=[X+Y-]=KXY/X+]aqueousorganicphase[Y-]inaqueousphase

[discussion:

relationshipbetweenDXandretentionvalue]

Ionpairchromatography（especiallyprimaryRP）solvestheproblemofseparatingthemixturebeforeitisdifficulttoseparate,suchasacid,alkaliandionicandnonionicmixtures,especiallytheseparationofsomebiochemicalsamplessuchasnucleicacids,nucleosides,alkaloidsanddrugsetc..

5.ionchromatography（Ion,Chromatography）

Theion-exchangeresinisusedasstationaryphase,andtheelectrolytesolutionismobilephase.Usingaconductivitydetectorasauniversaldetector,asuppressioncolumnwassetuptoeliminatetheinterferenceofthestrongelectrolytebackgroundionsinthemobilephaseontheconductivitydetector.Thereactionprincipleofthesamplecomponentontheseparatingcolumnandtherestrainingcolumnisthesameasthatoftheionexchangechromatography.

Takinganionexchangeresin（R-OH）asstationaryphaseandseparatinganions（suchasBr-）asanexample.WhentheanionBr-withthemobilephase（NaOH）entersthechromatographiccolumn,thefollowingexchangereactionoccurs（elutionisthereverseprocessoftheexchangereaction）:

Suppressreactionsonthecolumn:

r-h++na+,oh-===r-na++h2o

r-h++na+br-===r-na++h+br-------

可见,通过抑制柱将洗脱液转变成了电导值很小的水,消除了本底电导的影响;试样阴离子br-则被转化成了相应的酸h+br-,可用电导法灵敏的检测.

离子色谱法是溶液中阴离子分析的最佳方法.也可用于阳离子分析.

6.空间排阻色谱法（stericexclusionchromatography）

空间排阻色谱法以凝胶（gel）为固定相.它类似于分子筛的作用,但凝胶的孔径比分子筛要大得多,一般为数纳米到数百纳米.溶质在两相之间不是靠其相互作用力的不同来进行分离,而是按分子大小进行分离.分离只与凝胶的孔径分布和溶质的流动力学体积或分子大小有关.试样进入色谱柱后,随流动相在凝胶外部间隙以及孔穴旁流过.在试样中一些太大的分子不能进入胶孔而受到排阻,因此就直接通过柱子,首先在色谱图上出现,一些很小的分子可以进入所有胶孔并渗透到颗粒中,这些组分在柱上的保留值最大,在色谱图上最后出现.

高效液相色谱仪主要有进样系统、输液系统、.分离系统、检测系统和数据处理系统,下面将分别叙述其各自的组成与特点.

1.进样系统

一般采用隔膜注射进样器或高压进样间完成进样操作,进样量是恒定的.这对提高分析样品的重复性是有益的.

2.输液系统

该系统包括高压泵、流动相贮存器和梯度仪三部分.高压泵的一般压强为l.47～4.4x107pa,流速可调且稳定,当高压流动相通过层析柱时,可降低样品在柱中的扩散效应,可加快其在柱中的移动速度,这对提高分辨率、回收样品、保持样品的生物活性等都是有利的.流动相贮存错和梯度仪,可使流动相随固定相和样品的性质而改变,包括改变洗脱液的极性、离子强度、ph值,或改用竞争性抑制剂或变性剂等.这就可使各种物质（即使仅有一个基团的差别或是同分异构体）都能获得有效分离.

3.分离系统

该系统包括色谱柱、连接管和恒温器等.色谱柱一般长度为10～50cm（需要两根连用时,可在二者之间加一连接管）