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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,oneofwhichmovescontinuouslyinagivendirectionandinwhichtheindividualsubstancesexhibitdifferentmobilitiesbyreasonofdifferencesinadsorption,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。Thegeneralchromatographictechniquerequiresthatasoluteundergodistributionbetweentwophases,oneofthemfixed(stationaryphase),theothermoving(mobilephase).Itisthemobilephasethattransfersthesolutethroughthemediumuntiliteventuallyemergesseparatedfromothersolutesthatareelutedearlierorlater.Generally,thesoluteistransportedthroughtheseparationmediumbymeansofaflowingstreamofaliquidoragaseoussolventknownasthe“eluant.”Thestationaryphasemayactthroughadsorption,asinthecaseofadsorbentssuchasactivatedaluminaandsilicagel,oritmayactbydissolvingthesolute,thuspartitioningthelatterbetweenthestationaryandmobilephases.Inthelatterprocess,aliquidcoatedontoaninertsupport,orchemicallybondedontosilicagel,ordirectlyontothewallofafusedsilicacapillary,servesasthestationaryphase.Partitioningisthepredominantmechanismofseparationingas–liquidchromatography,paperchromatography,informsofcolumnchromatographyandinthin-layerchromatographydesignatedasliquid-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。ThetypesofchromatographyusefulinqualitativeandquantitativeanalysisthatareemployedintheUSPproceduresarecolumn,gas,paper,thin-layer,(includinghigh-performancethin-layerchromatography),andpressurizedliquidchromatography(commonlycalledhigh-pressureorhigh-performanceliquidchromatography).Paperandthin-layerchromatographyareordinarilymoreusefulforpurposesofidentification,becauseoftheirconvenienceandsimplicity.Columnchromatographyoffersawiderchoiceofstationaryphasesandisusefulfortheseparationofindividualcompounds,inquantity,frommixtures.Modernhigh-performancethin-layerchromatography,gaschromatography,andpressurizedliquidchromatographyrequiremoreelaborateapparatusbutusuallyprovidehighresolutionandidentifyandquantitateverysmallamountsofmaterial.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。UseofReferenceSubstancesinIdentityTests—Inpaperandthin-layerchromatography,theratioofthedistance(thisdistancebeingmeasuredtothepointofmaximumintensityofthespotorzone)traveledonthemediumbyagivencompoundtothedistancetraveledbythefrontofthemobilephase,fromthepointofapplicationofthetestsubstance,isdesignatedastheRFvalueofthecompound.TheratiobetweenthedistancestraveledbyagivencompoundandareferencesubstanceistheRRvalue.RFvaluesvarywiththeexperimentalconditions,andthusidentificationisbestaccomplishedwhereanauthenticspecimenofthecompoundinquestionisusedasareferencesubstanceonthesamechromatogram.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RR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-layeradsorbentoronthepaperinastraightline,paralleltotheedgeofthechromatographicplateorpaper,solutionsofthesubstancetobeidentified,theauthenticspecimen,andamixtureofnearlyequalamountsofthesubstancetobeidentifiedandtheauthenticspecimen.Eachsampleapplicationcontainsapproximatelythesamequantitybyweightofmaterialtobechromatographed.Ifthesubstancetobeidentifiedandtheauthenticspecimenareidentical,allchromatogramsagreeincolorandRFvalueandthemixedchromatogramyieldsasinglespot;i.e.,RRis1.0.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生了单个斑点；例如，RR为1.0。LocationandIdentificationofComponents—Thespotsproducedbypaperorthin-layerchromatographymaybelocatedby:(1)directinspectionifthecompoundsarevisibleunderwhiteoreithershort-wavelengt