美国药典USP3NF26色谱621(DOC74页)

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1、1/74 621 CHROMATOGRAPHY色谱法 INTRODUCTION 介绍 This chapter defines the terms and procedures used in chromatography and provides general information.Specific requirements for chromatographic procedures for drug substances and dosage forms,including adsorbent and developing solvents,are given in the indi

2、vidual monographs.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。Chromatography is defined as a procedure by which solutes are separated by a dynamic differential migration process in a system consisting of two or more phases,one of which moves continuously in a given direction a

3、nd in which the individual substances exhibit different mobilities by reason of differences in adsorption,partition,solubility,vapor pressure,molecular size,or ionic charge density.The individual substances thus separated can be identified or determined by analytical procedures.色谱法是应用溶质在两相或多相系统中的差速迁

4、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。The general chromatographic technique requires that a solute undergo distribution between two phases,one of them fixed(stationary phase),the other moving(mobile phase).It is the mobile phase that tran

5、sfers the solute through the medium until it eventually emerges separated from other solutes that are eluted earlier or later.Generally,the solute is transported through the separation medium by means of a flowing stream of a liquid or a gaseous solvent known as the“eluant.”The stationary phase may

6、act through adsorption,as in the case of adsorbents such as activated alumina and silica gel,or it may act by dissolving the solute,thus partitioning the latter between 2/74 the stationary and mobile phases.In the latter process,a liquid coated onto an inert support,or chemically bonded onto silica

7、gel,or directly onto the wall of a fused silica capillary,serves as the stationary phase.Partitioning is the predominant mechanism of separation in gasliquid chromatography,paper chromatography,in forms of column chromatography and in thin-layer chromatography designated as liquid-liquid chromatogra

8、phy.In practice,separations frequently result from a bination of adsorption and partitioning effects.Other separation principles include ion exchange,ion-pair formation,size exclusion,hydrophobic interaction,and chiral recognition.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

9、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 types of chromatogr

10、aphy useful in qualitative and quantitative analysis that are employed in the USP procedures are column,gas,paper,thin-layer,(including high-performance thin-layer chromatography),and pressurized liquid chromatography(monly called high-pressure or high-performance liquid chromatography).Paper and th

11、in-layer chromatography are ordinarily more useful for purposes of identification,because of their convenience and simplicity.Column chromatography offers a wider choice of stationary phases and is useful for the separation of individual pounds,in quantity,from mixtures.Modern high-performance thin-

12、layer chromatography,gas chromatography,and pressurized liquid chromatography require more elaborate apparatus but 3/74 usually provide high resolution and identify and quantitate very small amounts of material.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

13、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。Use of Reference Substances in Identity Tests In paper and thin-layer chromatography,the ratio of the distance(this distance being measured to the point of maximum intensity of the spot or

14、zone)traveled on the medium by a given pound to the distance traveled by the front of the mobile phase,from the point of application of the test substance,is designated as the RF value of the pound.The ratio between the distances traveled by a given pound and a reference substance is the RR value.RF

15、 values vary with the experimental conditions,and thus identification is best acplished where an authentic specimen of the pound in question is used as a reference substance on the same chromatogram.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

16、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。For this purpose,chromatograms are prepared by applying on the thin-layer adsorbent or on the paper in a straight line,parallel to the edge of the chromatographic plate or paper,solutions of the substance to be identified,the au

17、thentic specimen,and a mixture of nearly equal amounts of the substance to be identified and the authentic specimen.Each sample application contains approximately the same quantity by weight of material to be chromatographed.4/74 If the substance to be identified and the authentic specimen are ident

18、ical,all chromatograms agree in color and RF value and the mixed chromatogram yields a single spot;i.e.,RR is 1.0.为此，色谱图准备如下：在薄层吸附剂上或在纸 X 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。Location and

19、 Identification of ponents The spots produced by paper or thin-layer chromatography may be located by:(1)direct inspection if the pounds are visible under white or either short-wavelength(254 nm)or long-wavelength(360 nm)UV light,(2)inspection in white or UV light after treatment with reagents that

20、will make the spots visible(reagents are most conveniently applied with an atomizer),(3)use of a Geiger-Mller counter or autoradiographic techniques in the case of the presence of radioactive substances,or(4)evidence resulting from stimulation or inhibition of bacterial growth by the placing of remo

21、ved portions of the adsorbent and substance on inoculated media.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（360nm）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。In open-column chromatography,in pr

22、essurized liquid chromatography performed under conditions of constant flow rate,and in gas chromatography,the retention time,t,defined as the time elapsed between sample injection and appearance of the peak concentration of the eluted sample zone,may be used 5/74 as a parameter of identification.So

23、lutions of the substance to be identified or derivatives thereof,of the reference pound,and of a mixture of equal amounts of these two are chromatographed successively on the same column under the same chromatographic conditions.Only one peak should be observed for the mixture.The ratio of the reten

24、tion times of the test substance,the reference pound,and a mixture of these,to the retention time of an internal standard is called the relative retention time RR and is also used frequently as a parameter of identification.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，t

25、，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 RR，其也经常用于鉴别的参数。The deviations of RR,RF,or t values measured for the test substance from the values obtained for the reference pound and mixture should not exceed th

26、e reliability estimates determined statistically from replicate assays of the reference pound.从供试物质测得的 RR、RF、或 t 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。Chromatographic identification by these methods under given conditions strongly indicates identity but does not constitute definitiv

27、e identification.Coincidence of identity parameters under three to six different sets of chromatographic conditions(temperatures,column packings,adsorbents,eluants,developing solvents,various chemical derivatives,etc.)increases the probability that the test and reference substances are identical.How

28、ever,many isomeric pounds cannot be separated.Specific and pertinent chemical,6/74 spectroscopic,or physicochemical identification of the eluted ponent bined with chromatographic identity is the most valid criterion of identification.For this purpose,the individual ponents separated by chromatograph

29、y may be collected for further identification.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。PAPER CHROMATOGRAPHY 纸色谱法 In paper chromato

30、graphy the adsorbent is a sheet of paper of suitable texture and thickness.Chromatographic separation may proceed through the action of a single liquid phase in a process analogous to adsorption chromatography in columns.Since the natural water content of the paper,or selective imbibition of a hydro

31、philic ponent of the liquid phase by the paper fibers,may be regarded as a stationary phase,a partitioning mechanism may contribute significantly to the separation.在纸色谱法中吸附剂是适当质地与厚度的一 X 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,a

32、two-phase system may be used.The paper is impregnated with one of the phases,which then remains stationary(usually the more polar phase in the case of unmodified paper).The chromatogram is developed by slow passage of the other,mobile phase over the sheet.Development may be ascending,in which case t

33、he solvent is carried up the paper by capillary forces,7/74 or descending,in which case the solvent flow is also assisted by gravitational force.另外的选择是使用一个两相系统。这 X 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 X 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。D

34、ifferences in the value of RF have been reported where chromatograms developed in the direction of the paper grain(machine direction)are pared with others developed at right angles to the grain;therefore,the orientation of paper grain with respect to solvent flow should be maintained constant in a s

35、eries of chromatograms.(The machine direction is usually designated by the manufacturer on packages of chromatography paper.)有报道在将沿着纸 X 纹理方向（纤维方向）形成色谱图与沿着与纸 X 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，与溶剂流动有关的纸 X 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。）Descending Chromatography 下行色谱法 In descending chromato

36、graphy,the mobile phase flows downward on the chromatographic sheet.在下行层析法中，流动相在层析用纸上向下流动。Apparatus The essential equipment for descending chromatography consists of the following:仪器：用于下降层析法的基本设备有下列设备组成 A vapor-tight chamber provided with inlets for addition of solvent or for releasing internal pres

37、sure.The chamber is constructed preferably of glass,8/74 stainless steel,or porcelain and is so designed as to permit observation of the progress of the chromatographic run without opening of the chamber.Tall glass cylinders are convenient if they are made vapor-tight with suitable covers and a seal

38、ing pound.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。A rack of corrosion-resistant material about 5 cm shorter than the inside height of the chamber.The rack serves as a support for solvent troughs and for antisiphon rods which,in turn,hold up the chroma

39、tographic sheets.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。One or more glass troughs capable of holding a volume of solvent greater than that needed for one chromatographic run.The troughs must also be longer than the width of the chromatographic sheets.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

40、必须长于那些色谱纸的宽度。Heavy glass antisiphon rods to be supported by the rack and running outside of,parallel to,and slightly above the edge of the glass trough.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。Chromatographic sheets of special filter paper at least 2.5 cm wide and not wider than the length of the tro

41、ughs are cut to a length approximately equal to the height of the chamber.A fine pencil line is drawn horizontally across the filter paper at a distance from one end such that,when the sheet is suspended from the antisiphon rods with the upper end of the paper resting in the trough 9/74 and the lowe

42、r portion hanging free into the chamber,the line is located a few centimeters below the rods.Care is necessary to avoid contaminating the filter paper by excessive handling or by contact with dirty surfaces.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

43、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。Procedure The substance or substances to be analyzed are dissolved in a suitable solvent.Convenient volumes,delivered from suitable micropipets,of the resulting solution,normally containing 1 to 20 g of the pound,are placed in 6-to

44、 10-mm spots not less than 3 cm apart along the pencil line.If the total volume to be applied would produce spots of a diameter greater than 6 to 10 mm,it is applied in separate portions to the same spot,each portion being allowed to dry before the next is added.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

45、，其中通常含有 1 至 20g 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。The spotted chromatographic sheet is suspended in the chamber by use of the antisiphon rod,which holds the upper end of the sheet in the solvent trough.The bottom of the chamber is cover

46、ed with the prescribed solvent system.Saturation of the chamber with solvent vapor is facilitated by lining the inside walls with paper that is wetted with the prescribed solvent system.It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber

47、without touching the rack or the chamber walls or the fluid in the chamber.The chamber is sealed to allow equilibration(saturation)of the chamber and the paper with the solvent vapor.Any excess 10/74 pressure is released as necessary.For large chambers,equilibration overnight may be necessary.带斑点的色谱

48、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 X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。A volume of the mobile phase in excess of the volume required for plete development of the chromat

49、ogram is saturated with the immobile phase by shaking.After equilibration of the chamber,the prepared mobile solvent is introduced into the trough through the inlet.The inlet is closed and the mobile solvent phase is allowed to travel the desired distance down the paper.Precautions must be taken aga

50、inst allowing the solvent to run down the sheet when opening the chamber and removing the chromatogram.The location of the solvent front is quickly marked,and the sheets are dried.超过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

51、。迅速标注溶剂前端的位置，并干燥色谱纸。The chromatogram is observed and measured directly or after suitable development to reveal the location of the spots of the isolated drug or drugs.The paper section(s)predetermined to contain the isolated drug(s)may be cut out and eluted by an appropriate solvent,and the solution

52、s may be made up to a known volume and quantitatively analyzed by appropriate chemical or instrumental techniques.Similar procedures should be conducted with various amounts of similarly spotted reference standard on the same paper in the concentration range appropriate to prepare a valid calibratio

53、n curve.11/74 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 X 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。Ascending Chromatography 上行色谱法 In ascending chromatography,the lower edge of the sheet(or strip)is dipped into the

54、 mobile phase to permit the mobile phase to rise on the chromatographic sheet by capillary action.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。Apparatus The essential equipment for ascending chromatography is substantially the same as that described under Descending Chromatography.仪器：用于上行色谱法的基本设备实

55、质上与下行色谱法项下描述的相同。Procedure The test materials are applied to the chromatographic sheets as directed under Descending Chromatography,and above the level to which the paper is dipped into the developing solvent.The bottom of the developing chamber is covered with the developing solvent system.If a two-

56、phase system is used,both phases are added.It is also desirable to line the walls of the chamber with paper and to saturate this lining with the solvent system.Empty solvent troughs are placed on the bottom of the chamber,and the chromatographic sheets are suspended so that the end on which the spot

57、s have been added hangs free inside the empty trough.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，则两相均需加入。还期望完成的是，以纸 X 为该室的内壁加衬，并以溶剂系统浸透这12/74 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。The chamber is sealed,and equilibration is allowed to proceed as described under Descendin

58、g Chromatography.Then the developing solvent(mobile phase)is added through the inlet to the trough in excess of the solvent required for plete moistening of the chromatographic sheet.The chamber is resealed.When the solvent front has reached the desired height,the chamber is opened and the sheet is

59、removed and dried.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。Quantitative analyses of the spots may be conducted as described under Descending Chromatography.可以按照下行色谱法项下的描述对斑点进行定量分析。THIN-LAYER CHROMATOGRAPHY 薄层色谱法 在薄层色谱法中，吸附剂是相当薄的均匀涂层

60、，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 RF值完全相同且大小大致相等的斑点或区域。In thin-layer chromatography,the adsorbent is a relatively thin,uniform layer of dry,finely

61、powdered material applied to a glass,plastic,or metal sheet or plate,glass plates being most monly employed.The coated plate can be considered an“open chromatographic column”and the separations achieved may be 13/74 based upon adsorption,partition,or a bination of both effects,depending on the parti

62、cular type of stationary phase,its preparation,and its use with different solvents.Thin-layer chromatography on ion-exchange layers can be used for the fractionation of polar pounds.Presumptive identification can be effected by observation of spots or zones of identical RF value and about equal magn

63、itude obtained,respectively,with an unknown and a reference sample chromatographed on the same plate.A visual parison of the size or intensity of the spots or zones may serve for semiquantitative estimation.Quantitative measurements are possible by means of densitometry(absorbance or fluorescence me

64、asurements),or the spots may be carefully removed from the plate,followed by elution with a suitable solvent and spectrophotometric measurement.For two-dimensional thin-layer chromatography,the chromatographed plate is turned at a right angle and again chromatographed,usually in another chamber equi

65、librated with a different solvent system.对这些斑点或区域的大小或亮度进行检视可以作为半定量的评价。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。Apparatus Acceptable apparatus and materials for thin-layer chromatography consist of the following.仪器：薄层色谱法中可接受的仪

66、器和物料由下列组成：A TLC or HPTLC plate.The chromatography is generally carried out using precoated plates or sheets(on glass,aluminum,or polyester support)of suitable size.It may be necessary to clean the plates prior to separation.This can be done by migration of,or immersion in,an appropriate solvent.The plates may also be impregnated by procedures such as development,immersion,or spraying.At the time of use,the plates may be activated,if 14/74 necessary,by heating in an oven at 120 for 20 minutes.The