[工学]HPLC的方法及原理.pptVIP

妩媚人生 / High Performance Liquid Chromatography(HPLC) 妩媚人生 / High-performance Liquid Chromatography Early liquid chromatography was carried out in glass columns with diameters of 1 to 5 cm and lengths of 50 to 500 cm. Slow flow rates lead to very long separation times. Recently, column efficiency has been increased by making the packing material smaller and using very high pressure to force the mobile phase through the column. High-performance liquid chromatography is the general term used to distinguish the newer procedures from the basic methods. Advantages of HPLC Readily adaptable to accurate quantitative determinations. Suitable for separating nonvolatile species. Can separate thermally fragile species. Very applicable to separating substances of interest to industry, science, and the general public. Amino acids Proteins Nucleic acids Hydrocarbons Carbohydrates Drugs Terpenoids Pesticides Antibiotics Steroids Metal-organic species A variety of inorganic substances. HPLC的分类 吸附色谱 adsorption chromatography 分配色谱 partition chromatography 离子色谱 ion chromatography 分子排阻色谱 size exclusion chromatography 亲和色谱 affinity chromatography 吸附色谱的分离机理 分配色谱的分离机理 Chromatography Stationary Phases Normal vs. Reversed Phase Chromatography 离子色谱 离子交换色谱 ion exchange chromatography 离子对色谱 ion pair chromatography 离子排斥色谱 ion exclusion chromatography 离子对的分离机理 1.生成离子对－待测离子与离子对试剂生成中性““离子对”分布于 固定相与流动相之间，其分离类似传统的反相分离 2.动态离子交换－离子对试剂的疏水部分吸附于固定相形成动 态的离子交换表面，其分离机理类似于离子交换。 3.离子间相互作用－除包括以上两种分离机理和固定相表面双 电层结构的分离机理。 离子排斥色谱法的分离机理 1.Donnan排斥作用－Donnan膜的负电荷层排斥完全离解的离 子型化合物，仅允许未离解的化合物通过 2.吸附－保留时间与有机酸的烷基键的长度有关。通常烷基键越长，其保留时间也越长。 3. 空间排阻－与有机酸的分子量大小及交换树脂的交联度有关 分子排阻色谱的分离机理 可以分为 凝胶过滤 (gel filtration) 凝胶渗透 (gel permeation) Gel Filtration Chromatography: 流动相：为一般水溶液 固定相：为软质胶体，Agarose (sepharose)﹑