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1、English for Natural Products Chemistry,殷志琦 副教授,天然药化教研室,色谱法基本原理和分类Fundamental Principle and Classification of Chromatography,表1按两相所处状态分类 (state of two phase),stationary phase,表2按色谱原理分类,adsorption chromatography,gel chromatography,ion exchange chromatography,partition chromatography,affinity chromatog
2、raphy,表3按操作形式不同分类(operation form),column chromatography,thin layer chromatography,paper chromatography,membrane chromatography,1. Isolation and Chromatography 1.1 Paper Chromatography 1.2 Thin Layer Chromatography 1.3 High Performance Liquid Chromatography,1.1 纸色谱 Paper Chromatography (PC),PC,chroma
3、tography n. 色谱法，层析法chromatographic adj. 色谱法的，色谱仪的chromatogram n. 色谱，色谱图（谱）chromatograph n. 色谱仪 v. 用色谱法分析,PC,PC,Paper Chromatography,separation ： 分离、分析filter paper ：滤纸medium ：介质、媒介；培养基、媒介物reproducibility ：可重现性，再生性Rf value : 比移值 retardation factor(Rf)compound: 化合物 constituent 成分、构成 component 成分、组成的、构成
4、的anthocyanins ：花色素苷，花青苷pigments ：色素、颜料、染料,vocabulary,PC,1st paragraph,One of the main advantages of PC is the great convenience of carrying out separations simply on sheets of filter paper, which serve both as the medium for separation and as the support. Another advantage is the considerable reprod
5、ucibility of Rf values determined on paper, so that such measurements are valuable parameters for use in describing new plant compounds. Indeed, for substances such as the anthocyanins, which do not have other clearly defined physical properties, the Rf is the most important means of describing and
6、distinguishing the different pigments. the different pigments.,PC,1st paragraph,In search for bioactive constituents, we carried out chemical studies on Panax ginseng. We have systematically investigated the chemical constituents of Panax ginseng.,artition : n.分割，分配 v.分割，分裂，划分 partition chromatograp
7、hy 分配色谱 adsorption chromatography 吸附色谱water-immiscible 水不溶性的 alcoholic 醇的，乙醇的，酒精的 alcohol n. 酒精，乙醇solvent n. 溶剂n-butanol n. 正丁醇isopropanol n. 异丙醇t-butanol 叔丁醇,vocabulary,PC,n : normal 正（链）的iso: isotropic 异, 各向同性的, 同 t: tertiary 特, 叔, 第三,2nd paragraph,BAW ：n-butanol - acetic acid - water 4:1:5(上层) 正丁
8、醇 - 醋酸 - 水aqueous: 水的，水溶性的versatile：通用的,万能的,多用途的,多方面的 purines： 嘌呤pyrimidines： 嘧啶phenolic ：酚性的glycoside ： 苷，配糖物 glaiksaid,PC,vocabulary,glycose 单糖glycosidase 糖苷酶glaikusideisglycosidation 糖苷化glycosyl 糖基glaiksil,2nd paragraph,Chromatography on paper usually involves either partition or adsorption chrom
9、atography. In partition, the compounds are partitioned between a largely water-immiscible alcoholic solvent (e.g. n-butanol ) and water. The classic solvent mixture, n-butanol-acetic acid-water (4:1:5, top layer) (abbreviated as BAW) was indeed devised as a means of increasing the water content of t
10、he n-butanol layer and thus improving the utility of the solvent mixture.,PC,2nd paragraph,PC,2nd paragraph,Indeed, BAW is still widely applicable as a general solvent for many classed of plant constituent. By contrast, adsorption forces are one of the main features of PC in aqueous solvent. Pure wa
11、ter is a remarkably versatile chromato- graphic solvent and it can be used to separate the common purines and pyrimidines and is also applicable to phenolic compounds and to plant glycosides in general.,horizontal PC： 水平纸色谱circular PC：环形纸色谱TLC：Thin Layer Chromatography 薄层色谱resolution：分辨率carotenoids
12、：类胡萝卜素，类叶红素descending PC: 下行纸色谱法over-run：过度展开,PC,vocabulary,descending development 下行展开法ascending development 上行法two-dimensional development 二维分离,3rd paragraph,The choice of apparatus for PC depends to some extent on the amount of laboratory space available. Horizontal or circular PC, for example, t
13、akes up little more space than a standard TLC tank. It has remarkably good resolution and is used，for example, for separating carotenoids. In most laboratories, PC is carried out by descent, in tanks which will accommodate Whatman papers of the size 4657 cm. Descending PC is most useful since the so
14、lvent can be more easily over-run if this is desired; it is also slightly more convenient for two-dimensional separations.,PC,3rd paragraph,descending development,ascending development,two-dimensional development,cellulose ： 纤维素silicic acid ：硅酸 silica gel: 硅胶 silicon: 硅 silicone oil ：硅油alumina ：lju:
15、min 氧化铝，矾土lipids ：脂，类脂paraffin ：石蜡油，煤油 prfinreversed-phase chromatography：反相色谱milligram：毫克,deci(分) dm, centi(厘) cm, milli(毫) mm, mg, mlmicro(微) m, g, lnano(纳) nm, ng,PC,vocabulary,4th paragraph,A considerable range of modified filter papers are available commercially for achieving particular chromat
16、ographic separations. For example, the polar properties of cellulose can be reduced by in incorporating silicic acid or alumina into the papers, making them more suitable for separating lipids. Papers can likewise be modified in the laboratory, for example, by soaking them in paraffin or silicone oi
17、l in order to carry out reversed-phase chroma-tography, again for lipids. For large-scale separations, thick sheets of chromatography filter paper are available (Whatman no.3 or 3 MM) and these will cope with several milligrams of material per sheet.,PC,4th paragraph,UV-fluorescent：紫外荧光 UV: ultravio
18、let 紫外 fluorescent: 荧光 chromogenic reagent : 显色剂 chromogenic 发色的，发色体的diffusion ：扩散develop the colors：显色,PC,5th paragraph,vocabulary,developing agent (solvent) / tank/ time展开溶剂 / 缸 / 时间,development agent / rate/ system/ solution 展开溶剂 / 速度 / 系统 / 液,In PC, compounds are usually detected as coloured or
19、UV-fluorescent spots, after reaction with a chromogenic reagent, used either as a spray or as a dip. For large sheets, dipping is usually easier but the solvent content of the spray should be modified in order to facilitate quick drying and thus avoid diffusion during the dipping. The paper may then
20、 be heated in order to develop the colors.,PC,5th paragraph,solvent front： 溶剂前沿 origin： 原点fraction：分数；部分，馏份,PC,6th paragraph,vocabulary,The EtOH extract was subjected to silica gel column chromatography using CHCl3-CH3OH (9:1/5:5) as eluent to yield(afford) four fractions. The EtOAc extract was chro
21、matographed on silica gel eluting with CHCl3-CH3OH (9:1/5:5) to yield(afford) four fractions.,The Rf value is the distance a compound moves in chromatography relative to the solvent front. It is obtained by measuring the distance from the origin to the center of the spot produced by the substance, a
22、nd this is divided by the distance between the origin and the solvent front (i.e. the distance the solvent travels). This always appears as a fraction and lies between 0.01 and 0.99. It is convenient to multiply this by 100 and RF are quoted in this book as RFs (100). Elsewhere, RF (100) is sometime
23、s referred to as the hRF value.,PC,6th paragraph,When comparing RF values of a series of structurally related compounds. It is useful to refer to another chromatographic constant, the RMvalue. This is related to RF by the expression:,structurally related: 结构相关性的,PC,7th paragraph,1.2 薄层色谱 Thin Layer
24、Chromatography ( TLC),TLC,TLC：thin layer chromatography 薄层色谱sensitivity： n. 灵敏度adsorbent：吸附剂（物质） a. 能吸附的 adsorption： n. 吸附（作用）,TLC,vocabulary,silica gel：硅胶 celite： n. 硅藻土ion exchange resin： 离子交换树脂polyamide：聚酰胺 Sephadex：葡聚糖凝胶polyvinylpyrrolidone：聚乙烯吡咯烷酮 polyvinyl： n. a. 聚乙烯基化合物（的） pyrrolidone：吡咯烷酮,1t
25、h paragraph,aluminium 铝calcium 钙magnesium 镁sodium 钠,TLC,vocabulary,calcium hydroxide: 氢氧化钙calcium chloride： 氯化钙calcium oxide: 氧化钙calcium phosphate： 磷酸钙calcium sulphate： 硫酸钙,1th paragraph,The special advantages of TLC compared to PC include versatility, speed and sensitivity. Versatility is due to th
26、e fact that a number of different adsorbents besides cellulose may be spread on to a glass plate or other support and employed for chromatography. Although silica gel is most widely used, layers may be made up from aluminium oxide, celite, calcium hydroxide, ion exchange resin, magnesium phosphate,
27、polyamide, Sephadex, polyvinylpyrrolidone, cellulose and from mixtures of two or more of the above materials.,TLC,1th paragraph,The greater speed of TLC is due to the more compact nature of the adsorbent when spread on a plate and is an advantage when working with labile compounds. Finally, the sens
28、itivity of TLC is such that separations on less than g amounts of material can be achieved if necessary.,TLC,1th paragraph,spreading glass plates: 铺板automatic spreading devices：自动铺板器acetone：丙酮grease ： n. 油脂（状物）slurry ： n. 浆，泥浆hemihydrate：半水合物,TLC,vocabulary,etroleum ether ：石油醚 ether：乙醚chloroform：氯仿
29、ethyl acetate : 乙酸乙酯methanol：甲醇 ethanol：乙醇butanol ：正丁醇,2nd paragraph,air dried ： a. 风干的，晾干的activated： a. 活化的，活性的，激活的inorganic salt ：无机盐 silver nitrate 硝酸银 nitrate 硝酸根,TLC,vocabulary,inorganic： a. 无机的，无生物的 organic: a. 有机的，有机体的 inorganic acid / chemistry 无机酸/无机化学 organic acid/solvent/ chemistry 有机酸/有机
30、溶剂/有机化学,2nd paragraph,One of the original disadvantages of TLC was the labour of spreading glass plates with adsorbent, a labour somewhat eased by the later introduction of automatic spreading devices. Nevertheless, even with these, certain precautions are necessary.,TLC,2nd paragraph,The glass plat
31、es have to be carefully cleaned with acetone to remove grease. Then the slurry of silica gel (or other adsorbent) in water has to be vigorously shaken for a set time interval (e.g. 90s) before spreading. Depending on the particle size of the adsorbent, calcium sulphate hemihydrate (15%) may have to
32、be added to help bind the adsorbent on to the glass.,TLC,2nd paragraph,Finally, plates after spreading have to be air dried and then activated by heating in an oven at 100-110 for 30 min. In some separations, it is advantageous to modify the properties of the adsorbent by adding an inorganic salt (e
33、.g. silver nitrate for argentation TLC) and this is best done when the plate is being spread. Another reason for still using plates coated in the laboratory is that the moisture content of the silica gel can be controlled, a factor which is critical for some separations.,TLC,2nd paragraph,recoated p
34、lates ：预制板aluminium sheets ：铝片fluorescent indicator: 荧光指示剂quench the fluorescence：荧光淬灭wavelength：波长microparticles of silica：硅胶微粒HPLC：high performance liquid chromatography 高效液相色谱HPTLC：high performance thin layer chromatography 高效薄层色谱,TLC,vocabulary,3rd paragraph,Nowadays, however, it is usual to emp
35、loy precoated plates of commercial manufacture in most work, since these are more uniformand provide more reproducible results. Thereare a range of such plates available with different adsorbents, coated on glass, aluminium sheets or plastic. These may be with or without a fluorescent indicator, the
36、 addition of which allows the detection of all compounds which quench the fluorescence, when the plate is observed in UV light of 254 nm wavelength.,TLC,3rd paragraph,The most recent type of TLC plate is that coated with the same fine microparticles of silica that are used in the columns for HPLC. S
37、uch chromatography is termed HPTLC and it usually gives more efficient and rapid separations that on conventional silica layers.,TLC,3rd paragraph,solvent system：溶剂系统reference compounds ：参照（对照）化合物paper-lined：衬上滤纸saturated： a. 饱和的Horizontal TLC：水平薄层色谱over-run：过度展开electrophoresis：电泳,TLC,vocabulary,4th
38、 paragraph,A wider range of solvents have been applied to TLC than to PC and in general, there is more atitude in the exact proportions of different solvents used in a solvent system. RF values are considerably less reproducible than on paper and it is therefore essential to include one or more refe
39、rence compounds as markers. It is possible to standardize conditions for accurate measurement of RF in TLC, but this is a very tedious process.,TLC,4th paragraph,TLC is usually carried out by ascent, in a tank which is paper-lined so that the atmosphere inside is saturated with the solvent phase. Ho
40、rizontal TLC is employed，either when plates need to be over-run with solvent or when TLC is used in combination with electrophoresis.,TLC,4th paragraph,by spraying: 喷雾，喷洗conc. H2SO4：浓硫酸detection reagent：检测试剂steroids：甾体，甾族类化合物 steroidal: a.甾族的 sterol: n. 甾醇，固醇,TLC,vocabulary,sulphuric acid : 硫酸nitric
41、 acid ：硝酸hydrochloric acid：盐酸,5th paragraph,Detection of compounds on TLC plates in normally carried out by spraying, the smaller area of the plate (2020cm) making this a relatively simple procedure. One advantage over PC is that glass plates may be sprayed with conc. H2SO4, a useful detection reage
42、nt for steroids and lipids.,TLC,5th paragraph,reparative TLC: 制备薄层色谱 adsorbent： n. 吸附剂eluting：洗脱centrifuging：离心 ( centrifuge),TLC,vocabulary,elute: v. 洗脱（提），流出 elution: n. 洗脱，流出，淋洗 buffer elution 缓冲洗脱 gradient elution 梯度洗脱 isocratic elution 等度(无梯度)洗脱eluant: n. 洗脱（提）液，展开剂eluate: n. 洗脱液，提取液（物）,6th par
43、agraph,Preparative TLC is carried out using thick (up to 1mm) instead of thin (0.10-0.25mm) layers of adsorbent. Manufactured plates are available for this. Separated constituents are recovered by scraping off the adsorbent at the appropriate places on the developed plate, eluting the powder with a
44、solvent such as ether and finally centrifuging to remove the adsorbent.,TLC,6th paragraph,vocabulary,1.3 高效液相色谱High Performance Liquid Chromatography (HPLC),1 fluid reservior,2 pump,3 autosampler,3 sample injector,4 column(inside),4 column,1 fluid reservior,2 pump,5 detector I,3 autosampler,5 detect
45、or II,3 sample injector,4 column(inside),6 data processing system,chromatogram,Sample nameInjector volumeRun timeMethod name,HPLC,chromatogram,Sample nameRetention timeArea,HPLC,GLC：气相色谱 Gas liquid chromatographysensitivity:灵敏度、灵敏性quantitative: 定量的 qualitative: 定性的bond: 键，结合 bonded：键合的，化合的,vocabular
46、y,linearity and limit of detection (LOD) 线性和检测限precision（精密度） reproducibility（重复性）recovery（加样回收率） stability（稳定性）,HPLC,1st paragraph,stationary phase: 固定相octadecylsilyl silica gelmobile phase： 流动相miscible solvent mixture: 可互溶的混合溶剂,C18 or ODSC8silica gelSephadex ion exchange resin,methanol, acetonitri
47、le(乙腈) distilled wateracetic acidphosphoric acid(磷酸)buffer salt(缓冲盐),MeOH:H2O=85:15 CH3CN:H2O=70:30,vocabulary,HPLC,1st paragraph,olymer：聚合物 polymerase 聚合酶 hydrolase 水解酶 polymerization 聚合作用 decomposition 分解作用isocratic elution: 等度洗脱gradient elution: 梯度洗脱elute:洗脱, 流出,流出物 eluting:洗脱, 流出 elution:洗脱, 流出
48、elution time/ volume/program/ system,vocabulary,HPLC,1st paragraph,detector：检测器 UVD: ultraviolet-visible detector (紫外可见吸收检测器) PDAD: photodiode array detector (光电二极管阵列检测器) RID: refractive index detector (示差折光检测器） FD: fluorescence detector (荧光检测器) ECD: electrochemical detector (电化学检测器) ELSD: evaporati
49、ve light-scattering detector (蒸发光散射检测器）,vocabulary,HPLC,amino acid, vitamin, steroids, enzyme,lipids, sugars, amino acid, steroids, high polymers, saponin,1st paragraph,HPLC is analogous to GLC in its sensitivity and ability to provide both quantitative and qualitative data in the single operation.
50、It differs in that the stationary phase, bonded to a porous polymer is held in a narrow-bore stainless steel column and the liquid mobile phase is forced through under considerable pressure.,1st paragraph,HPLC,The apparatus for HPLC is more expensive than GLC, mainly because a suitable pumping syste
