1、精選優(yōu)質(zhì)文檔-傾情為你奉上精選優(yōu)質(zhì)文檔-傾情為你奉上專心-專注-專業(yè)專心-專注-專業(yè)精選優(yōu)質(zhì)文檔-傾情為你奉上專心-專注-專業(yè)621 CHROMATOGRAPHY色譜法 INTRODUCTION介紹 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, includ

2、ing adsorbent and developing solvents, are given in the individual monographs.此章節(jié)定義了色譜法中用到的術(shù)語和步驟，并提供了通用信息。對于原料藥和成藥的色譜步驟的具體要求，包括吸附劑和展開溶劑，在具體各論中給出。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 p

3、hases, one of which moves continuously in a given direction and 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 iden

4、tified or determined by analytical procedures.色譜法是應(yīng)用溶質(zhì)在兩相或多相系統(tǒng)中的差速遷移來進(jìn)行分離的技術(shù)，其中一相持續(xù)地向特定方向移動，而由于物質(zhì)在吸附性、分配、溶解性、氣體壓力、分子大小、或離子電荷密度上的差異，會顯示出不同的移動性。由此分開的這些單個物質(zhì)可以通過分析過程鑒別或測定。The general chromatographic technique requires that a solute undergo distribution between two phases, one of them fixed (stationary p

5、hase), the other moving (mobile phase). It is the mobile phase 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

6、liquid or a gaseous solvent known as the “eluant.” The stationary 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 proc

7、ess, a liquid coated onto an inert support, or chemically bonded 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 chrom

8、atography and in thin-layer chromatography designated as liquid-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,

9、and chiral recognition.常規(guī)色譜方法要求溶質(zhì)在兩相之間的分配，一個是固定的（固定相），另一個則在移動（移動相）。流動相的作用是穿過介質(zhì)轉(zhuǎn)移溶質(zhì)，直至其最終與其他不同時間洗脫出來的溶質(zhì)分開。通常，該溶質(zhì)由被稱為“洗脫劑”的某種液體或氣態(tài)溶劑的流動輸送著穿過分離介質(zhì)。該固定相可以通過吸附發(fā)揮作用，比如活性氧化鋁和硅膠之類的吸附劑，或者其可以通過溶解溶質(zhì)并由此在固定相與流動相之間分配溶質(zhì)來起作用。在后面這個過程中，涂在惰性載體上、或用化學(xué)方法鍵合在硅膠上、或直接在涂布石英毛細(xì)管壁上的某種液體作為固定相。分配作用是氣液色譜法、紙色譜法、多種柱色譜法和薄層色譜法稱為液-液色譜法中的

10、主要分離機(jī)制。在實際操作中，分離經(jīng)常是吸附與分配聯(lián)合作用的結(jié)果。其他分離原理包括離子交換、離子對結(jié)構(gòu)、空間排阻、疏水性相互作用、手性識別。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

11、chromatography (commonly called high-pressure or high-performance 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 i

12、s useful for the separation of individual compounds, in quantity, 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 am

13、ounts of material.在USP程序中使用的定性和定量分析中可以使用的色譜法類型是柱、氣相、薄層（包括高效薄層色譜法）、加壓液相色譜法（通常稱為高壓或高效液相色譜法）。由于方便、簡單，紙和薄層色譜法通常在鑒別用途中更加有效。柱色譜法對固定相提供了更廣泛的選擇，并且可用于從混合物中大量分離單個化合物?，F(xiàn)代高效薄層色譜法、氣相色譜法、加壓液相色譜法要求更加精密的儀器，通常提供高分離度，但只能識別與定量測定非常少量的物料。Use of Reference Substances in Identity Tests In paper and thin-layer chromatography

14、, the ratio of the distance (this distance being measured to the 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

15、 the compound. The ratio between the distances traveled by a given 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 substanc

16、e on the same chromatogram. 標(biāo)準(zhǔn)物質(zhì)在鑒別試驗中的使用：在紙和薄層色譜法中，從試樣點(diǎn)開始的某個特定化合物在介質(zhì)上的行進(jìn)距離（這個距離從斑點(diǎn)或者色帶的最深點(diǎn)測量）與流動相前端的行進(jìn)距離的比例被規(guī)定為該化合物的RF值。某個特定化合物和標(biāo)準(zhǔn)物質(zhì)的行進(jìn)距離之間的比值是RR值。RF值隨著試驗條件而變化，因此最好有待檢化合物可靠的標(biāo)準(zhǔn)品作為參考物質(zhì)并在同一色譜條件下完成鑒別試驗。For this purpose, chromatograms are prepared by applying on the thin-layer adsorbent or on the paper

17、in a straight line, parallel to the edge of the chromatographic 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

18、same quantity by weight of material to be chromatographed. If the 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.為此，色譜圖準(zhǔn)備如下：在薄層吸附劑上或在紙張上，在與色譜板或紙的底部邊緣平行的一條直線中，點(diǎn)上待識別物質(zhì)溶液

19、、其標(biāo)準(zhǔn)品溶液、以及由幾乎等量待識別物質(zhì)和其真實樣品構(gòu)成的混合物溶液。每個樣品點(diǎn)包含大約同樣重量的待層析物料。如果待識別物質(zhì)和其標(biāo)準(zhǔn)品是完全一樣的，則全部色譜圖在顏色和RF上會相符，且混合物的色譜圖產(chǎn)生了單個斑點(diǎn)；例如，RR為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

20、 either short-wavelength (254 nm) or long-wavelength (360 nm) UV 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 th

21、e case of the presence of radioactive substances, or (4) evidence resulting from stimulation or inhibition of bacterial growth by the placing of removed portions of the adsorbent and substance on inoculated media. 組分的位置與識別：由紙或薄層色譜法生成的斑點(diǎn)可以通過下面的方法定位 (1) 如果該化合物在可見光或者短波（254nm）或長波（360nm）紫外光下可見，直接檢查， (2)在

22、用能夠令斑點(diǎn)顯色的試劑處理之后（最方便的是用噴霧器噴灑試劑），在可見光或紫外光下檢查，(3)放射性物質(zhì)存在的情況下，使用蓋革-繆勒計數(shù)器或放射自顯影技術(shù)，或者(4)取出部分吸附劑和物質(zhì)置于接種介質(zhì)上，從細(xì)菌生長的促進(jìn)與抑制情況得到證據(jù)。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

23、 time elapsed between sample injection and appearance of the peak 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 c

24、hromatographed successively on the same column under the same chromatographic 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

25、the relative retention time RR and is also used frequently as a parameter of identification.在開放柱色譜中，在按照恒定流速條件進(jìn)行的加壓液相色譜法中，以及氣相色譜法中，被定義為在樣品進(jìn)樣與被洗脫樣品區(qū)域峰值濃度的出現(xiàn)之間所消耗時間的保留時間，t，可以用于鑒別的參數(shù)。待鑒別物質(zhì)或其衍生物的溶液、對照化合物的溶液、以及此二者含量相等的混合物的溶液須在相同的色譜條件下，使用同一個色譜柱進(jìn)行連續(xù)層析。只能在該混合物觀察到一個峰。供試物質(zhì)、對照化合物、以及二者的混合物的保留時間與內(nèi)標(biāo)物的保留時間的比值被稱為相對保

26、留時間RR，其也經(jīng)常用于鑒別的參數(shù)。The deviations of RR, RF, or t values measured 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.從供試物質(zhì)測得的RR、RF、或t值與從對照物質(zhì)和混合物中得到的

27、這些值之間的偏差不得超過從對照物質(zhì)重復(fù)含量測定中以統(tǒng)計學(xué)方法確定的可靠性評估值。Chromatographic identification 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 (temperatur

28、es, column packings, adsorbents, eluants, developing solvents, 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

29、identification of the eluted component combined with chromatographic identity is the most valid criterion of identification. For this purpose, the individual components separated by chromatography may be collected for further identification.在特定條件下以這些方法進(jìn)行的色譜鑒別有力地指明了對其的識別，但是不能構(gòu)成權(quán)威性的鑒別。識別參數(shù)在3至6套不同色譜條件（

30、溫度、柱填料、吸附劑、洗脫劑、展開劑、多種化學(xué)衍生物等）下均一致的情況增加了供試物質(zhì)和對照物質(zhì)完全相同的可能性。但是，很多同分異構(gòu)物無法分離。具體的相關(guān)化學(xué)、分光鏡檢查、或物理化學(xué)鑒別與色譜識別合在一起才是對于被洗脫組分的最有效的鑒別標(biāo)準(zhǔn)。為此，由色譜法分離的單個組分可以收集起來以便進(jìn)一步鑒別。PAPER CHROMATOGRAPHY紙色譜法 In paper chromatography the adsorbent is a sheet of paper of suitable texture and thickness. Chromatographic separation may pro

31、ceed 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 hydrophilic component of the liquid phase by the paper fibers, may be regarded as a stationary phase, a partitio

32、ning mechanism may contribute significantly to the separation.在紙色譜法中吸附劑是適當(dāng)質(zhì)地與厚度的一張紙。色譜分離可以在與柱中的吸附色譜法相似的工藝中，通過某單個液相的移動來進(jìn)行。因為紙含有天然的水分，或者紙纖維對于液相中親水組分的選擇性吸取，可以認(rèn)為是個固定相，所以分配機(jī)制可以對于分離作用明顯。Alternatively, a two-phase system may be used. The paper is impregnated with one of the phases, which then remains stati

33、onary (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 the solvent is carried up the paper by capillary forces, or descending, in which case the solvent

34、 flow is also assisted by gravitational force.另外的選擇是使用一個兩相系統(tǒng)。這張紙浸漬在其中一個相中，然后其保持不動（如果使用未改性紙，通常選擇極性較大的相）。通過將另一個相，即流動相，緩慢穿過這張紙來使色譜圖形成。色譜圖的形成過程可以是上行的，這樣溶劑被毛細(xì)管作用力支撐著沿著紙向上，這個過程也可以是下行的，在此情況下溶劑流動也受到重力的影響。Differences in the value of RF have been reported where chromatograms developed in the direction of the

35、paper grain (machine direction) are compared 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 series of chromatograms. (The machine direction is usually designated by the manufacturer on pack

36、ages of chromatography paper.)有報道在將沿著紙張紋理方向（纖維方向）形成色譜圖與沿著與紙張紋理呈直角方向形成的色譜圖進(jìn)行比較之后，RF值有一定的差異,因此，與溶劑流動有關(guān)的紙張紋理定向應(yīng)該在一系列色譜圖中保持恒定。（纖維方向通常由制造商在色譜紙的包裝上標(biāo)出。）Descending Chromatography下行色譜法 In descending chromatography, the mobile phase flows downward on the chromatographic sheet.在下行層析法中，流動相在層析用紙上向下流動。Apparatus T

37、he essential equipment for descending chromatography consists of the following: 儀器：用于下降層析法的基本設(shè)備有下列設(shè)備組成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 designe

38、d 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 sealing compound.裝有添加溶劑或釋放內(nèi)部壓力的入口的氣密室。該室最好以玻璃、或不銹鋼、或瓷構(gòu)成，且設(shè)計為不用打開該室即可觀察層析運(yùn)行的進(jìn)展。如果以適當(dāng)?shù)纳w子和密封物確保其密閉，高玻璃園筒即可使用。

39、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 chromatographic sheets.短于該室內(nèi)部高度5cm的耐腐蝕材料支架。該支架作為用于溶劑槽以及用于抗虹吸棒的支撐，這些抗虹吸棒依次撐起色譜紙。One or more glass troug

40、hs 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.一個或多個能夠容納多于一次色譜運(yùn)行溶劑需要量的玻璃槽。該槽也必須長于那些色譜紙的寬度。Heavy glass antisiphon rods to be supported by the rack and running outside of, para

41、llel to, and slightly above the edge of the glass trough.玻璃抗虹吸棒將由支架支撐并在玻璃槽邊緣之外、與邊緣平行、略微高于邊緣的位置放置。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 i

42、s 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 and the lower portion hanging free into the chamber, the line is located a few centimeters below the rods. Car

43、e is necessary to avoid contaminating the filter paper by excessive handling or by contact with dirty surfaces.將至少2.5cm寬并且寬度不超過玻璃槽長度的特殊濾紙的色譜紙切至長度約等于氣密室高度。距離濾紙的一端一段距離，畫一道水平細(xì)鉛筆線，距離的選擇應(yīng)確保當(dāng)此色譜紙從抗虹吸棒上懸掛下來，并且該紙上端擱在玻璃槽中而下邊的部分自由地在氣密室中垂下的時候，該鉛筆線位于玻璃棒下面幾厘米。必須小心防止過度處理或與骯臟表面接觸而污染濾紙。Procedure The substance or su

44、bstances 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 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 ap

45、plied 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. 步驟：待分析的一個或多個物質(zhì)溶解于適當(dāng)溶劑中。以微量吸管吸取適當(dāng)體積的溶液，其中通常含有1至20g該化合物，沿著鉛筆線6至10mm的大小斑點(diǎn)間的間隔不小于3cm。如果將要點(diǎn)樣的總體積會產(chǎn)生直徑超過6至10mm的斑點(diǎn)，則將其分段點(diǎn)于同一個斑點(diǎn)，在

46、同一位置點(diǎn)樣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 covered with the prescribed solvent system. Saturation of the chamber with solvent vapor is facilitat

47、ed 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 without touching the rack or the chamber walls or the fluid in the chamber. The chamber is sea

48、led to allow equilibration (saturation) of the chamber and the paper with the solvent vapor. Any excess pressure is released as necessary. For large chambers, equilibration overnight may be necessary.帶斑點(diǎn)的色譜紙以抗虹吸棒懸掛在氣密室內(nèi)，該棒將該色譜紙的上端固定在溶劑槽中。氣密室的底部以規(guī)定的溶劑系統(tǒng)覆蓋。使用以規(guī)定溶劑系統(tǒng)潤濕的紙張襯托于氣密室的內(nèi)壁，以增加氣密室的溶劑蒸汽飽和度。重要的是要確

49、保色譜紙掛在抗虹吸棒下的部分自由的懸掛在氣密室中，沒有接觸到支架、或室壁、或室內(nèi)的液體。氣密室被密閉，以便使該室與色譜紙達(dá)到溶劑蒸汽平衡（飽和）。在需要時，釋放任何多余壓力。對于大氣密室而言，可能必須過夜以達(dá)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, t

50、he 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 allowing the solvent to run down the sheet when opening the chamber and removing th

51、e chromatogram. The location of the solvent front is quickly marked, and the sheets are dried.超過色譜圖完全形成所必需量的流動相通過振搖與固定相飽和。在該室達(dá)到平衡之后，配制好的流動溶劑通過入口加入到玻璃槽中。關(guān)閉入口，且讓流動溶劑相沿著色譜紙向下行進(jìn)需要的距離。必須采取預(yù)防措施，在打開氣密室并取出色譜圖時，防止溶劑沿色譜紙向下流動。迅速標(biāo)注溶劑前端的位置，并干燥色譜紙。The chromatogram is observed and measured directly or after suitab

52、le 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 solutions may be made up to a known volume and quantitatively analyzed by appropriate chem

53、ical 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 calibration curve.直接或用適當(dāng)措施顯示被分離出來的一個或多個藥物的斑點(diǎn)位置之后，觀察并測量該色譜圖?？梢詫⑸V紙上預(yù)計含有分離出的（多種）藥物的（多個）部分切下，并

54、用適當(dāng)?shù)娜軇┫疵摚梢灾瞥啥噙_(dá)某個已知體積的溶液并以適當(dāng)?shù)幕瘜W(xué)或者儀器方法進(jìn)行定量分析。使用適于建立有效校正曲線的濃度范圍內(nèi)的不同數(shù)量的標(biāo)準(zhǔn)品，以相同方法在同樣的色譜紙上點(diǎn)樣，并進(jìn)行相同的步驟。Ascending Chromatography 上行色譜法In ascending chromatography, the lower edge of the sheet (or strip) is dipped into the mobile phase to permit the mobile phase to rise on the chromatographic sheet by capill

55、ary action.在上行色譜法中，色譜紙（或色譜帶）底端浸入流動相中，以使流動相通過毛細(xì)管作用力在色譜紙上上升。Apparatus The essential equipment for ascending chromatography is substantially the same as that described under Descending Chromatography. 儀器：用于上行色譜法的基本設(shè)備實質(zhì)上與下行色譜法項下描述的相同。Procedure The test materials are applied to the chromatographic sheets

56、 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-phase system is used, both phases are added. It is also desirable to line the walls

57、 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 spots have been added hangs free inside the empty trough. 步驟：按照下行色譜法中的規(guī)定將供試品點(diǎn)于色譜紙上，

58、位置高于色譜紙浸在展開溶劑中的水平。展開室底部平鋪以展開溶劑系統(tǒng)。如果使用兩相系統(tǒng)，則兩相均需加入。還期望完成的是，以紙張為該室的內(nèi)壁加襯，并以溶劑系統(tǒng)浸透這層內(nèi)壁。將空容積槽置于該室的底部，并將色譜紙懸掛起來并使已經(jīng)加上斑點(diǎn)的一端自由吊在空槽內(nèi)。The chamber is sealed, and equilibration is allowed to proceed as described under Descending Chromatography. Then the developing solvent (mobile phase) is added through the inl

59、et to the trough in excess of the solvent required for complete 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 removed and dried.該室需封閉，并使之達(dá)到平衡，以按照下行色譜法項下描述的進(jìn)行。然后，通過入口將展開溶劑（流動相）加入至槽內(nèi)，數(shù)量需超過

60、徹底潤濕色譜紙所必須的數(shù)量。將該室重新封閉。當(dāng)溶劑面到達(dá)需要的高度之后，打開該室，取出該色譜紙并干燥。Quantitative analyses of the spots may be conducted as described under Descending Chromatography. 可以按照下行色譜法項下的描述對斑點(diǎn)進(jìn)行定量分析。THIN-LAYER CHROMATOGRAPHY薄層色譜法 在薄層色譜法中，吸附劑是相當(dāng)薄的均勻涂層，以干燥、細(xì)粉狀物料涂于玻璃、塑料、或金屬薄片或薄板，玻璃薄板是最常用的。帶涂層的薄板能夠被認(rèn)為是一個“開放色譜柱”而且所實現(xiàn)的分離可以是基于吸收、分配