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1、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 individ
2、ual 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 and
3、 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
4、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。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
5、that transfers 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 stationa
6、ry phase may 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 the stationary and mobile phases. In the latter process, a liquid coated onto an inert support, or chemically bonde
7、d onto silica 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
8、-liquid chromatography. In practice, separations frequently result from a combination 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
9、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
10、、手性识别。The types of chromatography 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 (commonly called high-pressure or high-performan
11、ce liquid chromatography). Paper and thin-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 compounds, in quantit
12、y, from mixtures. Modern high-performance thin-layer chromatography, gas chromatography, and pressurized liquid chromatography require more elaborate apparatus but 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
13、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。Use of Reference Substances in Identity Tests In paper and thin-layer chromatography, the ratio of the distance (this distance being measured to th
14、e point of maximum intensity of the spot or zone) traveled on the medium by a given compound 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 compound. The ratio between the distances traveled by a gi
15、ven compound and a reference substance is the RR value. RF values vary with the experimental conditions, and thus identification is best accomplished where an authentic specimen of the compound 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
16、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。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
17、 plate or paper, solutions of the substance to be identified, the authentic 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. If t
18、he substance to be identified and the authentic specimen are identical, 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
19、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 Identification of Components The spots produced by paper or thin-layer chromatography may be located by: (1) direct inspection if the compounds are visible under white or either short-wavelength (254 nm) or long-wavelength (360 nm) U
20、V light, (2) inspection in white or UV light after treatment with reagents that 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) eviden
21、ce resulting from stimulation or inhibition of bacterial growth by the placing of removed 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-
22、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 pressurized 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 pe
23、ak concentration of the eluted sample zone, may be used as a parameter of identification. Solutions of the substance to be identified or derivatives thereof, of the reference compound, and of a mixture of equal amounts of these two are chromatographed successively on the same column under the same c
24、hromatographic conditions. Only one peak should be observed for the mixture. The ratio of the retention times of the test substance, the reference compound, 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
25、 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，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 measure
26、d for the test substance from the values obtained for the reference compound and mixture should not exceed the reliability estimates determined statistically from replicate assays of the reference compound.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 identif
27、ication by these methods under given conditions strongly indicates identity but does not constitute definitive identification. Coincidence of identity parameters under three to six different sets of chromatographic conditions (temperatures, column packings, adsorbents, eluants, developing solvents,
28、various chemical derivatives, etc.) increases the probability that the test and reference substances are identical. However, many isomeric compounds cannot be separated. Specific and pertinent chemical, spectroscopic, or physicochemical identification of the eluted component combined with chromatogr
29、aphic identity is the most valid criterion of identification. For this purpose, the individual components separated by chromatography 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
30、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 chromatography 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 a
31、nalogous to adsorption chromatography in columns. Since the natural water content of the paper, or selective imbibition of a hydrophilic component 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
32、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, a 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 p
33、aper). The chromatogram is developed by slow passage of the other, mobile phase over the sheet. Development may be ascending, in which case the solvent is carried up the paper by capillary forces, 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。这张
34、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。Differences in the value of RF have been reported where chromatograms developed in the direction of the paper grain (machine direction) are compared with others develo
35、ped at right angles to the grain; therefore, the orientation of paper grain with respect to solvent flow should be maintained constant in a series of chromatograms. (The machine direction is usually designated by the manufacturer on packages of chromatography paper.)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
36、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。）Descending Chromatography下行色谱法 In descending chromatography, 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 o
37、f 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 pressure. The chamber is constructed preferably of glass, stainless steel, or porcelain and is so designed as to permit observation of the progress of the chromatograph
38、ic run without opening of the chamber. Tall glass cylinders are convenient if they are made vapor-tight with suitable covers and a sealing compound.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
39、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 chromatographic 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 nee
40、ded 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
41、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 troughs 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
42、 one end such that, when the sheet is suspended from the antisiphon rods with the upper end of the paper resting in the trough and the lower 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 exces
43、sive 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. Co
44、nvenient volumes, delivered from suitable micropipets, of the resulting solution, normally containing 1 to 20 g of the compound, are placed in 6- to 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
45、, 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，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
46、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 covered 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 th
47、e prescribed solvent system. It is important to ensure that the portion of the sheet hanging below the rods is freely suspended in the chamber 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
48、paper with the solvent vapor. Any excess pressure is released as necessary. For large chambers, equilibration overnight may be necessary.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
49、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 complete development of the chromatogram 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 against allo
