USP31-621色谱法 中文译稿

1、621CHROMATOGRAPHY色谱法INTRODUCTION介绍Thischapterdefinesthetermsandproceduresusedinchromatographyandprovidesgeneralinformation.Specificrequirementsforchromatographicproceduresfordrugsubstancesanddosageforms,includingadsorbentanddevelopingsolvents,aregivenintheindividualmonographs.此章节定义了色谱法中用到的术语和步骤，并提供了

2、通用信息。对于原料药和成药的色谱步骤的具体要求，包括吸附剂和展开溶剂，在具体各论中给出。Chromatographyisdefinedasaprocedurebywhichsolutesareseparatedbyadynamicdifferentialmigrationprocessinasystemconsistingoftwoormorephases,oneofwhichmovescontinuouslyinagivendirectionandinwhichtheindividualsubstancesexhibitdifferentmobilitiesbyreasonofdiffere

3、ncesinadsorption,partition,solubility,vaporpressure,molecularsize,orionicchargedensity.Theindividualsubstancesthusseparatedcanbeidentifiedordeterminedbyanalyticalprocedures.色谱法是应用溶质在两相或多相系统中的差速迁移来进行分离的技术，其中一相持续地向特定方向移动，而由于物质在吸附性、分配、溶解性、气体压力、分子大小、或离子电荷密度上的差异，会显示出不同的移动性。由此分开的这些单个物质可以通过分析过程鉴别或测定。Thegen

4、eralchromatographictechniquerequiresthatasoluteundergodistributionbetweentwophases,oneofthemfixed(stationaryphase),theothermoving(mobilephase).Itisthemobilephasethattransfersthesolutethroughthemediumuntiliteventuallyemergesseparatedfromothersolutesthatareelutedearlierorlater.Generally,thesoluteistra

5、nsportedthroughtheseparationmediumbymeansofaflowingstreamofaliquidoragaseoussolventknownasthe“eluant.”Thestationaryphasemayactthroughadsorption,asinthecaseofadsorbentssuchasactivatedaluminaandsilicagel,oritmayactbydissolvingthesolute,thuspartitioningthelatterbetweenthestationaryandmobilephases.Inthe

6、latterprocess,aliquidcoatedontoaninertsupport,orchemicallybondedontosilicagel,ordirectlyontothewallofafusedsilicacapillary,servesasthestationaryphase.Partitioningisthepredominantmechanismofseparationingas-quidchromatography,paperchromatography,informsofcolumnchromatographyandinthin-layerchromatograp

7、hydesignatedasliquid-liquidchromatography.Inpractice,separationsfrequentlyresultfromacombinationofadsorptionandpartitioningeffects.Otherseparationprinciplesincludeionexchange,ion-pairformation,sizeexclusion,hydrophobicinteraction,andchiralrecognition.常规色谱方法要求溶质在两相之间的分配，一个是固定的(固定相)，另一个则在移动(移动相)。流动相的作

8、用是穿过介质转移溶质，直至其最终与其他不同时间洗脱出来的溶质分开。通常，该溶质由被称为“洗脱剂”的某种液体或气态溶剂的流动输送着穿过分离介质。该固定相可以通过吸附发挥作用，比如活性氧化铝和硅胶之类的吸附剂，或者其可以通过溶解溶质并由此在固定相与流动相之间分配溶质来起作用。在后面这个过程中，涂在惰性载体上、或用化学方法键合在硅胶上、或直接在涂布石英毛细管壁上的某种液体作为固定相。分配作用是气液色谱法、纸色谱法、多种柱色谱法和薄层色谱法称为液-液色谱法中的主要分离机制。在实际操作中，分离经常是吸附与分配联合作用的结果。其他分离原理包括离子交换、离子对结构、空间排阻、疏水性相互作用、手性识别。The

9、typesofchromatographyusefulinqualitativeandquantitativeanalysisthatareemployedintheUSPproceduresarecolumn,gas,paper,thin-layer,(includinghigh-performancethin-layerchromatography),andpressurizedliquidchromatography(commonlycalledhigh-pressureorhigh-performanceliquidchromatography).Paperandthin-layerc

10、hromatographyareordinarilymoreusefulforpurposesofidentification,becauseoftheirconvenienceandsimplicity.Columnchromatographyoffersawiderchoiceofstationaryphasesandisusefulfortheseparationofindividualcompounds,inquantity,frommixtures.Modernhigh-performancethin-layerchromatography,gaschromatography,and

11、pressurizedliquidchromatographyrequiremoreelaborateapparatusbutusuallyprovidehighresolutionandidentifyandquantitateverysmallamountsofmaterial.在USP程序中使用的定性和定量分析中可以使用的色谱法类型是柱、气相、薄层（包括高效薄层色谱法）、加压液相色谱法（通常称为高压或高效液相色谱法）。由于方便、简单，纸和薄层色谱法通常在鉴别用途中更加有效。柱色谱法对固定相提供了更广泛的选择，并且可用于从混合物中大量分离单个化合物。现代高效薄层色谱法、气相色谱法、加压液相

12、色谱法要求更加精密的仪器，通常提供高分离度，但只能识别与定量测定非常少量的物料。UseofReferenceSubstancesinIdentityTestsInpaperandthin-layerchromatography,theratioofthedistance（thisdistancebeingmeasuredtothepointofmaximumintensityofthespotorzone）traveledonthemediumbyagivencompoundtothedistancetraveledbythefrontofthemobilephase,fromthepoint

13、ofapplicationofthetestsubstance,isdesignatedastheRvalueofFthecompound.TheratiobetweenthedistancestraveledbyagivencompoundandareferencesubstanceistheRvalue.RvaluesvarywiththeRFexperimentalconditions,andthusidentificationisbestaccomplishedwhereanauthenticspecimenofthecompoundinquestionisusedasareferen

14、cesubstanceonthesamechromatogram.标准物质在鉴别试验中的使用：在纸和薄层色谱法中，从试样点开始的某个特定化合物在介质上的行进距离（这个距离从斑点或者色带的最深点测量）与流动相前端的行进距离的比例被规定为该化合物的Rf值。某个特定化合物和标准物质的行进距离之间的比值是值。Rf值随着试验条件而变化，因此最好有待检化合物可靠的标准品作为参考物质并在同一色谱条件下完成鉴别试验。Forthispurpose,chromatogramsarepreparedbyapplyingonthethin-layeradsorbentoronthepaperinastraightli

15、ne,paralleltotheedgeofthechromatographicplateorpaper,solutionsofthesubstancetobeidentified,theauthenticspecimen,andamixtureofnearlyequalamountsofthesubstancetobeidentifiedandtheauthenticspecimen.Eachsampleapplicationcontainsapproximatelythesamequantitybyweightofmaterialtobechromatographed.Ifthesubst

16、ancetobeidentifiedandtheauthenticspecimenareidentical,allchromatogramsagreeincolorandRvalueandthemixedchromatogramFyieldsasinglespot;i.e.,Ris1.0.R为此，色谱图准备如下：在薄层吸附剂上或在纸张上，在与色谱板或纸的底部边缘平行的一条直线中，点上待识别物质溶液、其标准品溶液、以及由几乎等量待识别物质和其真实样品构成的混合物溶液。每个样品点包含大约同样重量的待层析物料。如果待识别物质和其标准品是完全一样的，则全部色谱图在颜色和RF上会相符，且混合物的色谱图产

17、生了单个斑点；例如，Rr为1.00LocationandIdentificationofComponentsThespotsproducedbypaperorthin-layerchromatographymaybelocatedby:(1)directinspectionifthecompoundsarevisibleunderwhiteoreithershort-wavelength(254nm)orlong-wavelength(360nm)UVlight,(2)inspectioninwhiteorUVlightaftertreatmentwithreagentsthatwillmak

18、ethespotsvisible(reagentsaremostconvenientlyappliedwithanatomizer),(3)useofaGeiger-Mullercounterorautoradiographictechniquesinthecaseofthepresenceofradioactivesubstances,or(4)evidenceresultingfromstimulationorinhibitionofbacterialgrowthbytheplacingofremovedportionsoftheadsorbentandsubstanceoninocula

19、tedmedia.组分的位置与识别：由纸或薄层色谱法生成的斑点可以通过下面的方法定位(1)如果该化合物在可见光或者短波(254nm)或长波(36Onm)紫外光下可见，直接检查，(2)在用能够令斑点显色的试剂处理之后(最方便的是用喷雾器喷洒试剂)，在可见光或紫外光下检查，(3)放射性物质存在的情况下，使用盖革-缪勒计数器或放射自显影技术，或者(4)取出部分吸附剂和物质置于接种介质上，从细菌生长的促进与抑制情况得到证据。Inopen-columnchromatography,inpressurizedliquidchromatographyperformedunderconditionsofcon

20、stantflowrate,andingaschromatography,theretentiontime,t,definedasthetimeelapsedbetweensampleinjectionandappearanceofthepeakconcentrationoftheelutedsamplezone,maybeusedasaparameterofidentification.Solutionsofthesubstancetobeidentifiedorderivativesthereof,ofthereferencecompound,andofamixtureofequalamo

21、untsofthesetwoarechromatographedsuccessivelyonthesamecolumnunderthesamechromatographicconditions.Onlyonepeakshouldbeobservedforthemixture.Theratiooftheretentiontimesofthetestsubstance,thereferencecompound,andamixtureofthese,totheretentiontimeofaninternalstandardiscalledtherelativeretentiontimeRandis

22、alsousedRfrequentlyasaparameterofidentification.在开放柱色谱中，在按照恒定流速条件进行的加压液相色谱法中，以及气相色谱法中，被定义为在样品进样与被洗脱样品区域峰值浓度的出现之间所消耗时间的保留时间，r,可以用于鉴别的参数。待鉴别物质或其衍生物的溶液、对照化合物的溶液、以及此二者含量相等的混合物的溶液须在相同的色谱条件下，使用同一个色谱柱进行连续层析。只能在该混合物观察到一个峰。供试物质、对照化合物、以及二者的混合物的保留时间与内标物的保留时间的比值被称为相对保留时间只型其也经常用于鉴别的参数。ThedeviationsofR,R,orrvalue

23、smeasuredforthetestsubstancefromtheRFvaluesobtainedforthereferencecompoundandmixtureshouldnotexceedthereliabilityestimatesdeterminedstatisticallyfromreplicateassaysofthereferencecompound.从供试物质测得的rr、只尸或r值与从对照物质和混合物中得到的这些值之间的偏差不得超过从对照物质重复含量测定中以统计学方法确定的可靠性评估值。Chromatographicidentificationbythesemethods

24、undergivenconditionsstronglyindicatesidentitybutdoesnotconstitutedefinitiveidentification.Coincidenceofidentityparametersunderthreetosixdifferentsetsofchromatographicconditions(temperatures,columnpackings,adsorbents,eluants,developingsolvents,variouschemicalderivatives,etc.)increasestheprobabilityth

25、atthetestandreferencesubstancesareidentical.However,manyisomericcompoundscannotbeseparated.Specificandpertinentchemical,spectroscopic,orphysicochemicalidentificationoftheelutedcomponentcombinedwithchromatographicidentityisthemostvalidcriterionofidentification.Forthispurpose,theindividualcomponentsse

26、paratedbychromatographymaybecollectedforfurtheridentification.在特定条件下以这些方法进行的色谱鉴别有力地指明了对其的识别，但是不能构成权威性的鉴别。识别参数在3至6套不同色谱条件(温度、柱填料、吸附剂、洗脱剂、展开剂、多种化学衍生物等)下均一致的情况增加了供试物质和对照物质完全相同的可能性。但是，很多同分异构物无法分离。具体的相关化学、分光镜检查、或物理化学鉴别与色谱识别合在一起才是对于被洗脱组分的最有效的鉴别标准。为此，由色谱法分离的单个组分可以收集起来以便进一步鉴别。PAPERCHROMATOGRAPHY纸色谱法Inpaperc

27、hromatographytheadsorbentisasheetofpaperofsuitabletextureandthickness.Chromatographicseparationmayproceedthroughtheactionofasingleliquidphaseinaprocessanalogoustoadsorptionchromatographyincolumns.Sincethenaturalwatercontentofthepaper,orselectiveimbibitionofahydrophiliccomponentoftheliquidphasebythep

28、aperfibers,mayberegardedasastationaryphase,apartitioningmechanismmaycontributesignificantlytotheseparation.在纸色谱法中吸附剂是适当质地与厚度的一张纸。色谱分离可以在与柱中的吸附色谱法相似的工艺中，通过某单个液相的移动来进行。因为纸含有天然的水分，或者纸纤维对于液相中亲水组分的选择性吸取，可以认为是个固定相，所以分配机制可以对于分离作用明显。Alternatively,atwo-phasesystemmaybeused.Thepaperisimpregnatedwithoneoftheph

29、ases,whichthenremainsstationary(usuallythemorepolarphaseinthecaseofunmodifiedpaper).Thechromatogramisdevelopedbyslowpassageoftheother,mobilephaseoverthesheet.Developmentmaybeascending,inwhichcasethesolventiscarriedupthepaperbycapillaryforces,ordescending,inwhichcasethesolventflowisalsoassistedbygrav

30、itationalforce.另外的选择是使用一个两相系统。这张纸浸渍在其中一个相中，然后其保持不动（如果使用未改性纸，通常选择极性较大的相）。通过将另一个相，即流动相，缓慢穿过这张纸来使色谱图形成。色谱图的形成过程可以是上行的，这样溶剂被毛细管作用力支撑着沿着纸向上，这个过程也可以是下行的，在此情况下溶剂流动也受到重力的影响。DifferencesinthevalueofRFhavebeenreportedwherechromatogramsdevelopedinthedirectionofthepapergrain（machinedirection）arecomparedwithother

31、sdevelopedatrightanglestothegrain;therefore,theorientationofpapergrainwithrespecttosolventflowshouldbemaintainedconstantinaseriesofchromatograms.（Themachinedirectionisusuallydesignatedbythemanufactureronpackagesofchromatographypaper.）有报道在将沿着纸张纹理方向（纤维方向）形成色谱图与沿着与纸张纹理呈直角方向形成的色谱图进行比较之后，Rf值有一定的差异，因此，与溶剂

32、流动有关的纸张纹理定向应该在一系列色谱图中保持恒定。（纤维方向通常由制造商在色谱纸的包装上标出。）DescendingChromatography下行色谱法Indescendingchromatography,themobilephaseflowsdownwardonthechromatographicsheet.在下行层析法中，流动相在层析用纸上向下流动。ApparatusTheessentialequipmentfordescendingchromatographyconsistsofthefollowing:仪器：用于下降层析法的基本设备有下列设备组成Avapor-tightchambe

33、rprovidedwithinletsforadditionofsolventorforreleasinginternalpressure.Thechamberisconstructedpreferablyofglass,stainlesssteel,orporcelainandissodesignedastopermitobservationoftheprogressofthechromatographicrunwithoutopeningofthechamber.Tallglasscylindersareconvenientiftheyaremadevapor-tightwithsuita

34、blecoversandasealingcompound.装有添加溶剂或释放内部压力的入口的气密室。该室最好以玻璃、或不锈钢、或瓷构成，且设计为不用打开该室即可观察层析运行的进展。如果以适当的盖子和密封物确保其密闭，高玻璃园筒即可使用。Arackofcorrosion-resistantmaterialabout5cmshorterthantheinsideheightofthechamber.Therackservesasasupportforsolventtroughsandforantisiphonrodswhich,inturn,holdupthechromatographicshee

35、ts.短于该室内部高度5cm的耐腐蚀材料支架。该支架作为用于溶剂槽以及用于抗虹吸棒的支撑，这些抗虹吸棒依次撑起色谱纸。Oneormoreglasstroughscapableofholdingavolumeofsolventgreaterthanthatneededforonechromatographicrun.Thetroughsmustalsobelongerthanthewidthofthechromatographicsheets.一个或多个能够容纳多于一次色谱运行溶剂需要量的玻璃槽。该槽也必须长于那些色谱纸的宽度。Heavyglassantisiphonrodstobesuppor

36、tedbytherackandrunningoutsideof,parallelto,andslightlyabovetheedgeoftheglasstrough.玻璃抗虹吸棒将由支架支撑并在玻璃槽边缘之外、与边缘平行、略微高于边缘的位置放置。Chromatographicsheetsofspecialfilterpaperatleast2.5cmwideandnotwiderthanthelengthofthetroughsarecuttoalengthapproximatelyequaltotheheightofthechamber.Afinepencillineisdrawnhoriz

37、ontallyacrossthefilterpaperatadistancefromoneendsuchthat,whenthesheetissuspendedfromtheantisiphonrodswiththeupperendofthepaperrestinginthetroughandthelowerportionhangingfreeintothechamber,thelineislocatedafewcentimetersbelowtherods.Careisnecessarytoavoidcontaminatingthefilterpaperbyexcessivehandling

38、orbycontactwithdirtysurfaces.将至少2.5cm宽并且宽度不超过玻璃槽长度的特殊滤纸的色谱纸切至长度约等于气密室高度。距离滤纸的一端一段距离，画一道水平细铅笔线，距离的选择应确保当此色谱纸从抗虹吸棒上悬挂下来，并且该纸上端搁在玻璃槽中而下边的部分自由地在气密室中垂下的时候，该铅笔线位于玻璃棒下面几厘米。必须小心防止过度处理或与肮脏表面接触而污染滤纸。ProcedureThesubstanceorsubstancestobeanalyzedaredissolvedinasuitablesolvent.Convenientvolumes,deliveredfromsuit

39、ablemicropipets,oftheresultingsolution,normallycontaining1to20pgofthecompound,areplacedin6-to10-mmspotsnotlessthan3cmapartalongthepencilline.Ifthetotalvolumetobeappliedwouldproducespotsofadiametergreaterthan6to10mm,itisappliedinseparateportionstothesamespot,eachportionbeingallowedtodrybeforethenexti

40、sadded.步骤：待分析的一个或多个物质溶解于适当溶剂中。以微量吸管吸取适当体积的溶液，其中通常含有1至20pg该化合物，沿着铅笔线6至10mm的大小斑点间的间隔不小于3cm。如果将要点样的总体积会产生直径超过6至10mm的斑点，则将其分段点于同一个斑点，在同一位置点样之前的部分放置至干。Thespottedchromatographicsheetissuspendedinthechamberbyuseoftheantisiphonrod,whichholdstheupperendofthesheetinthesolventtrough.Thebottomofthechamberiscove

41、redwiththeprescribedsolventsystem.Saturationofthechamberwithsolventvaporisfacilitatedbyliningtheinsidewallswithpaperthatiswettedwiththeprescribedsolventsystem.Itisimportanttoensurethattheportionofthesheethangingbelowtherodsisfreelysuspendedinthechamberwithouttouchingtherackorthechamberwallsortheflui

42、dinthechamber.Thechamberissealedtoallowequilibration(saturation)ofthechamberandthepaperwiththesolventvapor.Anyexcesspressureisreleasedasnecessary.Forlargechambers,equilibrationovernightmaybenecessary.带斑点的色谱纸以抗虹吸棒悬挂在气密室内，该棒将该色谱纸的上端固定在溶剂槽中。气密室的底部以规定的溶剂系统覆盖。使用以规定溶剂系统润湿的纸张衬托于气密室的内壁，以增加气密室的溶剂蒸汽饱和度。重要的是要确

43、保色谱纸挂在抗虹吸棒下的部分自由的悬挂在气密室中，没有接触到支架、或室壁、或室内的液体。气密室被密闭，以便使该室与色谱纸达到溶剂蒸汽平衡(饱和)。在需要时，释放任何多余压力。对于大气密室而言，可能必须过夜以达平衡。Avolumeofthemobilephaseinexcessofthevolumerequiredforcompletedevelopmentofthechromatogramissaturatedwiththeimmobilephasebyshaking.Afterequilibrationofthechamber,thepreparedmobilesolventisintrod

44、ucedintothetroughthroughtheinlet.Theinletisclosedandthemobilesolventphaseisallowedtotravelthedesireddistancedownthepaper.Precautionsmustbetakenagainstallowingthesolventtorundownthesheetwhenopeningthechamberandremovingthechromatogram.Thelocationofthesolventfrontisquicklymarked,andthesheetsaredried.超过

45、色谱图完全形成所必需量的流动相通过振摇与固定相饱和。在该室达到平衡之后，配制好的流动溶剂通过入口加入到玻璃槽中。关闭入口，且让流动溶剂相沿着色谱纸向下行进需要的距离。必须采取预防措施，在打开气密室并取出色谱图时，防止溶剂沿色谱纸向下流动。迅速标注溶剂前端的位置，并干燥色谱纸。Thechromatogramisobservedandmeasureddirectlyoraftersuitabledevelopmenttorevealthelocationofthespotsoftheisolateddrugordrugs.Thepapersection(s)predeterminedtoconta

46、intheisolateddrug(s)maybecutoutandelutedbyanappropriatesolvent,andthesolutionsmaybemadeuptoaknownvolumeandquantitativelyanalyzedbyappropriatechemicalorinstrumentaltechniques.Similarproceduresshouldbeconductedwithvariousamountsofsimilarlyspottedreferencestandardonthesamepaperintheconcentrationrangeap

47、propriatetoprepareavalidcalibrationcurve.直接或用适当措施显示被分离出来的一个或多个药物的斑点位置之后，观察并测量该色谱图。可以将色谱纸上预计含有分离出的（多种）药物的（多个）部分切下，并用适当的溶剂洗脱，可以制成多达某个已知体积的溶液并以适当的化学或者仪器方法进行定量分析。使用适于建立有效校正曲线的浓度范围内的不同数量的标准品，以相同方法在同样的色谱纸上点样，并进行相同的步骤。AscendingChromatography上行色谱法Inascendingchromatography,theloweredgeofthesheet（orstrip）isdi

48、ppedintothemobilephasetopermitthemobilephasetoriseonthechromatographicsheetbycapillaryaction.在上行色谱法中，色谱纸（或色谱带）底端浸入流动相中，以使流动相通过毛细管作用力在色谱纸上上升。ApparatusTheessentialequipmentforascendingchromatographyissubstantiallythesameasthatdescribedunderDescendingChromatography.仪器：用于上行色谱法的基本设备实质上与下行色谱法项下描述的相同。Proce

49、dureThetestmaterialsareappliedtothechromatographicsheetsasdirectedunderDescendingChromatography,andabovetheleveltowhichthepaperisdippedintothedevelopingsolvent.Thebottomofthedevelopingchamberiscoveredwiththedevelopingsolventsystem.Ifatwo-phasesystemisused,bothphasesareadded.Itisalsodesirabletolineth

50、ewallsofthechamberwithpaperandtosaturatethisliningwiththesolventsystem.Emptysolventtroughsareplacedonthebottomofthechamber,andthechromatographicsheetsaresuspendedsothattheendonwhichthespotshavebeenaddedhangsfreeinsidetheemptytrough.步骤：按照下行色谱法中的规定将供试品点于色谱纸上，位置高于色谱纸浸在展开溶剂中的水平。展开室底部平铺以展开溶剂系统。如果使用两相系统，则

51、两相均需加入。还期望完成的是，以纸张为该室的内壁加衬，并以溶剂系统浸透这层内壁。将空容积槽置于该室的底部，并将色谱纸悬挂起来并使已经加上斑点的一端自由吊在空槽内。Thechamberissealed,andequilibrationisallowedtoproceedasdescribedunderDescendingChromatography.Thenthedevelopingsolvent(mobilephase)isaddedthroughtheinlettothetroughinexcessofthesolventrequiredforcompletemoisteningofthec

52、hromatographicsheet.Thechamberisresealed.Whenthesolventfronthasreachedthedesiredheight,thechamberisopenedandthesheetisremovedanddried.该室需封闭，并使之达到平衡，以按照下行色谱法项下描述的进行。然后，通过入口将展开溶剂(流动相)加入至槽内，数量需超过彻底润湿色谱纸所必须的数量。将该室重新封闭。当溶剂面到达需要的高度之后，打开该室，取出该色谱纸并干燥。Quantitativeanalysesofthespotsmaybeconductedasdescribedun

53、derDescendingChromatography.可以按照下行色谱法项下的描述对斑点进行定量分析。THIN-LAYERCHROMATOGRAPHY薄层色谱法在薄层色谱法中，吸附剂是相当薄的均匀涂层，以干燥、细粉状物料涂于玻璃、塑料、或金属薄片或薄板，玻璃薄板是最常用的。带涂层的薄板能够被认为是一个“开放色谱柱”而且所实现的分离可以是基于吸收、分配，或两种效果的联合作用，取决于固定相的特定种类、其配制方法、不同溶剂的应用。在离子交换层上的薄层色谱法能够用于极性化合物的分离。推定鉴别能够这样实现，分别使用未知样品和标准物质样品在同一块薄板上进行层析，并观察所得结果中只尸值完全相同且大小大致相

54、等的斑点或区域。Inthin-layerchromatography,theadsorbentisarelativelythin,uniformlayerofdry,finelypowderedmaterialappliedtoaglass,plastic,ormetalsheetorplate,glassplatesbeingmostcommonlyemployed.Thecoatedplatecanbeconsideredan“openchromatographiccolumn”andtheseparationsachievedmaybebaseduponadsorption,partit

55、ion,oracombinationofbotheffects,dependingontheparticulartypeofstationaryphase,itspreparation,anditsusewithdifferentsolvents.Thin-layerchromatographyonion-exchangelayerscanbeusedforthefractionationofpolarcompounds.PresumptiveidentificationcanbeeffectedbyobservationofspotsorzonesofidenticalRFvalueanda

56、boutequalmagnitudeobtained,respectively,withanunknownandareferencesamplechromatographedonthesameplate.Avisualcomparisonofthesizeorintensityofthespotsorzonesmayserveforsemiquantitativeestimation.Quantitativemeasurementsarepossiblebymeansofdensitometry(absorbanceorfluorescencemeasurements),orthespotsm

57、aybecarefullyremovedfromtheplate,followedbyelutionwithasuitablesolventandspectrophotometricmeasurement.Fortwo-dimensionalthin-layerchromatography,thechromatographedplateisturnedatarightangleandagainchromatographed,usuallyinanotherchamberequilibratedwithadifferentsolventsystem.对这些斑点或区域的大小或亮度进行检视可以作为半

58、定量的评价。定量测定可以通过光密度分析法(吸光率或荧光测量法)，或者可以将这些斑点仔细地从薄板上移走，并使用适当的溶剂进行洗脱并用分光光度法测定。对于双向薄层色谱法，将点样后的薄板转一个直角，并通常在以不同溶剂系统平衡后的另一个室中，再次展开。ApparatusAcceptableapparatusandmaterialsforthin-layerchromatographyconsistofthefollowing.仪器：薄层色谱法中可接受的仪器和物料由下列组成：ATLCorHPTLCplate.Thechromatographyisgenerallycarriedoutusingpreco

59、atedplatesorsheets(onglass,aluminum,orpolyestersupport)ofsuitablesize.Itmaybenecessarytocleantheplatespriortoseparation.Thiscanbedonebymigrationof,orimmersionin,anappropriatesolvent.Theplatesmayalsobeimpregnatedbyproceduressuchasdevelopment,immersion,orspraying.Atthetimeofuse,theplatesmaybeactivated

60、,ifnecessary,byheatinginanovenat120for20minutes.ThestationaryphaseofTLCplateshasanaverageparticlesizeof10-5pm,andthatofHPTLCplatesanaverageparticlesizeof5pm.Commercialplateswithapreadsorbantzonecanbeusediftheyarespecifiedinamonograph.Sampleappliedtothepreabsorbantregiondevelopsintosharp,narrowbandsa