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1、高效液相 色谱分析 High Performance Liquid Chromatography,简介 基本原理 特点 仪器流程及主要部件 分离模式 应用与发展动向,High-performance liquid chromatography (HPLC) is a form of liquid chromatography to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a se
2、paration column, and a detector.,Introduction,Compounds are separated by injecting a plug of the sample mixture onto the column.,Understanding how a chromatographic column works - Bands,Introduction,The different components in the mixture pass through the column at different rates due to differences
3、 in their partitioning behavior between the mobile liquid phase and the stationary phase.,Introduction,The HPLC System,GC system,气相色谱仪由以下五大系统组成：气路系统、进样系统、分离系统、温控系统、检测记录系统。,HPLC system,气相色谱仪由以下五大系统组成：液路系统（梯度冲洗装置） 、进样系统、分离系统、温控系统、检测记录系统。,HPLC system,高效液相色谱是在气相色谱和经典色谱的基础上发展起来的。 不同点仅仅是现代液相色谱比经典液相色谱有较高的效
4、率和实现了自动化操作。 经典的液相色谱法：常压，固定相柱效低，分析周期长。 现代液相色谱法引用了气相色谱的理论，流动相改为高压输送（最高输送压力可达4.9107Pa）；色谱柱是以特殊的方法用小粒径的填料填充而成，从而使柱效大大高于经典液相色谱（每米塔板数可达几万或几十万）；同时柱后连有高灵敏度的检测器，可对流出物进行连续检测。 因此，高效液相色谱具有分析速度快、分离效能高、自动化等特点。所以人们称它为高压、高速、高效或现代液相色谱法。,HPLC system - Principle,液相色谱法的分离机理是基于混合物中各组分对两相亲和力的差别。,分配系数的差异,反相 (reverse phase
5、, RP) 正相 (normal phase, NP),等度 (isocratic) 梯度 (gradient),定性分析 (qualitative analysis) 定量分析 (quantitative analysis),分析型 (analytical HPLC) 半制备型 (semi-preparative HPLC) 制备型 (preparative HPLC) 工程级 (engineering level),液相系统操作,高效液相色谱量级,分析目的,色谱柱,HPLC system,- Solvents,化合物的极性是由分子中所含官能团的种类、数目及排列方式等综合因素决定。 常见能团
6、极性强弱顺序是：RCOOH ArOH HOH ROH NHC=OCH3 RNH2 RSH RCHO RCOR RCOOR RN(Me)2 RNO2 ROCH3 CH=CH CH2CH2,利用不同组分与固定相（固定化分子）的高专属性亲和力进行分离。常用于蛋白质的分离。,色谱法,吸附色谱法,利用组分在固定相（吸附剂）上的物理吸附能力的差异而达到分离的方法,分配色谱法,利用组分在固定相（固定液）中溶解度不同而达到分离的方法,离子交换色谱法 (IEC),利用组分在固定相（离子交换剂）上的亲和力大小不同而达到分离的方法,凝胶色谱法,(空间排阻色谱法),利用多孔固定相对不同大小的分子的排阻作用达到分离的方
7、法。包括凝胶过滤色谱（GFC）和凝胶渗透色谱（GPC）,手性色谱法,利用化合物的旋光特性达到分离的方法,亲和色谱法,分 离 机 理,分离机理,色谱法,吸附色谱法,离子交换色谱法,吸附剂选择,硅胶 酸性物质 氧化铝 碱性物质 聚酰胺 可与酚类/酸类等形成氢键 活性炭 分离水溶性成分,洗脱剂选择,二元溶剂 三元溶剂,阳离子交换树脂 -SO3H, -PO3H2, -COOH, Ac-OH,阴离子交换树脂 -NH2, =NH, -N+(CH3)3OH-,酸、碱、盐的水溶液或缓冲溶液,凝胶色谱法 (GPC),(空间排阻色谱法),离子交换色谱法的应用,用于中药有效部位的分离（不同电荷离子的分离）,2. 用
8、于同类型天然有机物的组分分离（相同电荷离子的分离）,3. 提纯抗生素,4. 提取分离生物碱,5. 分离纯化糖类化合物,6. 分离纯化核苷酸及核酸,7. 提取精制胆红素,Cation Exchange Chromatography (CEC),Anion Exchange Chromatography (AEC),阴离子交换色谱,阳离子交换色谱,http:/ Characterization,Electrophoresis,Protein Characterization,Isoelectric Focusing,Protein Characterization,Two-Dimensional
9、Electrophoresis (2D-GEL),Protein Characterization,http:/ (UV-vis) 折光指数检测器 光电二极管阵列检测器 荧光检测器 质谱检测器 (LC-MS) 电化学检测器 ,HPLC system - Detector,Quality Control,When you are handling Active Pharmaceutical Ingredients which will be used for clinical trials, it is most important to have dedicated and very reli
10、able personnel in quality control. Regulations for the analysis of APIs increase year by year so it is our privilege to have an experienced and ambitious team eager to solve highly challenging analytical problems. The quality control unit not only possesses a large variety of the latest analytical i
11、nstruments but has also quick and unlimited access to the vast resources of analytical equipment of the University of Zurich.,http:/www.lpf.uzh.ch/quality_control.html,http:/www.lpf.uzh.ch/quality_control.html,3 HPLC instruments for routine analysis and method development 1 HPLC instrument for chira
12、l applications only 1 GC-FID instrument for routine analysis 1 HPLC/MS instrument(Ion Trap) for mass identification and structure elucidation (MS/MS) 1 Headspace-GC instrument for residual solvent analysis 1 FT-IR instrument with ATR measurement of solids, liquids, pastes, etc. 1 DSC instrument Full
13、y equipped laboratory for the analysis of highly active compounds,Method development for all types of HPLC and GC analysis Full palette of EP/USP analysis Purity and assay determination Impurity profile characterization Release of chemicals for GMP production and final products (APIs) Certificates o
14、f Analysis Long term stability studies Structure elucidation Thermochemical analysis Karl Fischer titration for water Combustion elemental analysis Residue on ignition Residual solvent analysis Analysis of highly active compounds,QC Services,QC Equipment,Solid-Phase Extraction (SPE),http:/ is actual
15、ly column liquid-solid chromatography. Since SPE is liquid chromatography LC, its practice is governed by LC principles. SPE Sample Enrichment and Purification using SPE,Illustration of a general elution protocol for normal-phase chromatography on SPE cartridges (Silica, Florisil, Alumina, Diol, CN, NH2),http:/ of a general elution protocol for reversed-phase chro
