色谱基本原理.ppt

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1、现代色谱分析现代色谱分析胡坪化学与分子工程学院分析教研组T学习资源1.书籍书籍 气相色谱（毛细管气相色谱）气相色谱（毛细管气相色谱）液相色谱、液相色谱、毛细管电泳毛细管电泳 平面色谱、平面色谱、离子色谱、凝胶色谱离子色谱、凝胶色谱 色谱联用技术色谱联用技术 制备色谱、逆流色谱等制备色谱、逆流色谱等 色谱技术在各领域的应用色谱技术在各领域的应用2.杂志杂志J CHROMATOGR A 3.756J CHROMATOGR B 2.5ADV CHROMATOGR 2.6BIOMED CHROMATOGR 1.592CHROMATOGRAPHIA 1.312J CHROMATOGR SCI 1.135

2、J LIQ CHROMATOGR R T 1.026色谱（中文核心期刊）色谱（中文核心期刊）3.网站，小木虫，丁香园等，小木虫，丁香园等4.仪器厂商Agilent，Waters,岛津，Varian等教学内容和安排（教学内容和安排（24学时）学时）1.色谱理论基础（色谱理论基础（5学时）学时）2.气相色谱法（气相色谱法（3学时）学时）3.液相色谱法（液相色谱法（4学时）学时）4.色谱相关技术（色谱相关技术（2学时）学时）5.毛细管电泳（毛细管电泳（1学时）学时）6.超临界流体色谱法（超临界流体色谱法（1学时）学时）7.制备色谱（制备色谱（1学时）学时）8.高速逆流色谱法（高速逆流色谱法（1学时）

3、学时）9.色谱联用技术（色谱联用技术（4学时）学时）10.多维色谱（多维色谱（2学时）学时）实验安排（实验安排（5选选2，24学学时时）1.气相色谱分离条件优化气相色谱分离条件优化2.毛细管气相色谱法在复杂样品分析的应用毛细管气相色谱法在复杂样品分析的应用3.高效液相色谱条件优化高效液相色谱条件优化4.气质联用（演示实验）气质联用（演示实验）5.离子色谱离子色谱（演示实验）（演示实验）实验时间另排实验时间另排要求撰写实验报告，记为平时成绩（要求撰写实验报告，记为平时成绩（20%20%）开卷考试，开卷考试，80%80%Chapter 1 Principle of Chromatography色谱

4、理论基础色谱理论基础 色谱分离基本原理色谱分离基本原理 色谱热力学理论色谱热力学理论 色谱动力学理论色谱动力学理论 色谱分离条件优化理论色谱分离条件优化理论 色谱定性和定量方法色谱定性和定量方法1 Basic principle of chromatographic separation1.1 色谱分离基本原理色谱分离基本原理Partition coefficient K分配系数1.2 Types of chromatography 色谱分类色谱分类According to the forms of stationary phase:nPlanar chromatography，平面色谱，平面

5、色谱-Thin Layer chromatography(TLC)，薄，薄层色谱层色谱-Paper chromatography(PC)，纸色谱，纸色谱nColumn chromatography，柱色谱，柱色谱According to the form of mobile phase:-Gas chromatography(GC)，气相色谱，气相色谱-Liquid chromatography(LC)，液相色谱，液相色谱-Supercritical fluid chromatography(SFC)，超临界流体色谱，超临界流体色谱nAccording to the principle of

6、separationAdsorption chromatography，吸附色谱，吸附色谱Partition chromatography，分配色谱，分配色谱Ion exchange chromatography，离子交换色谱，离子交换色谱Molecular exclusion chromatography，凝胶色谱，凝胶色谱Affinity chromatography，亲和色谱，亲和色谱1.3 Instrument 色谱仪色谱仪流动相流动相进样装置进样装置色谱柱色谱柱检测器检测器数据记录数据记录与处理与处理Chromatogram应用举例2.Thermodynamics of chroma

7、tography色谱热力学理论色谱热力学理论 2.1 The language of chromatography术语2.1.1 About the chromatogrampeakPeak heightBase linePeak widthPeak width at half heightPeak areaStandard error:峰峰高基线峰宽半峰宽峰面积标准偏差Tailing peak，拖尾峰，拖尾峰Leading peak，伸舌峰，伸舌峰Ghost peak，鬼峰，鬼峰Distorted peak，畸峰，畸峰Baseline draft，基线漂移，基线漂移Baseline nois

8、e，基线噪声，基线噪声Band broadening，谱带扩展，谱带扩展Peak capacity，峰容量，峰容量2.1.2 About retention values Retention time 保留时间保留时间 Dead time 死时间死时间 Adjusted retention time 调整保留时间调整保留时间 Retention volume 保留体积保留体积 Dead volume 死体积死体积 Adjusted retention volume 调整保留体积调整保留体积nCorrected retention volume 校正保留体积nNet retention volu

9、me 净保留体积nSpecific retention time 比保留体积nRelative retention time 相对保留时间nRetention index 保留指数2.2 Thermodynamic property of retention values 保留值的热力学属性保留值的热力学属性（以气液色谱法为例）（以气液色谱法为例）2.2.1 Retention values and distribution coefficientnThree kinds of expression on K分配系数的三种表达 Temp.of column Temp.of column 0C

10、nThe relationship between retention values and KEssence of retention value is thermodynamic.保留值的本质是物质的热力学性质保留值的本质是物质的热力学性质 Two conceptsCapacity fact容量因子 Phase ratio相比2.2.2 The thermodynamic equation of retention value保留值的热力学方程保留值的热力学方程 Ideal solution:Raoults lawIdeal gas state equation:Herry law:Con

11、clusion:1.It is suitable for chromatography to separate the homologue.气相色谱方法适合分离同系物气相色谱方法适合分离同系物2.Changing stationary phase is the efficient method to improve the separation.改变固定相的种类是提高分离的有效途径改变固定相的种类是提高分离的有效途径3.The effect of temperature?温度如何影响分离？温度如何影响分离？2.2.3 The effect of temperature on retention

12、 value 温度对保留值的影响温度对保留值的影响提高柱温，保留值减小提高柱温，保留值减小改变柱温可能改变出峰顺序改变柱温可能改变出峰顺序3.Dynamics of chromatography色谱动力学nStudy on the essential of band broadening（谱带展宽的本质）nStudy of the effects on the band broadening（影响谱带展宽的因素）Two important theoriesnPlate theory塔板理论nRate theory速率理论3.1 塔板理论塔板理论导出色谱流出曲线方程导出色谱流出曲线方程Theor

13、etical plate 理论塔板数理论塔板数Height equivalent of a theoretical plate 塔板高度塔板高度 H=L/n3.2 速率理论速率理论1956,Van Deemter Study on the effects on the band broadening by using the rules of fluid molecular.Educe the relationship between height equivalent of a theoretical plate and linearity flow rate of mobile phase

14、.研究色谱峰展宽的原因研究色谱峰展宽的原因Van deemters equation:H=A+B/u+CuA：涡流扩散项，：涡流扩散项，由不等路径造成的色谱由不等路径造成的色谱峰展宽峰展宽A=2dp涡流扩散项均匀性因子粒径B：分子扩散项，：分子扩散项，由于分子的纵向扩散造由于分子的纵向扩散造成的色谱峰展宽成的色谱峰展宽nB/unB=2Dg弯曲因子扩散系数C：传质阻力项，由质量交换时间（传质阻：传质阻力项，由质量交换时间（传质阻力）所造成的色谱峰展宽力）所造成的色谱峰展宽n（Cg+Cl）un Cg=n Cl=容量因子液膜厚度液相扩散系数气相色谱中的速率方程气相色谱中的速率方程What can w

15、e learn from the equation?nResearch and development of types of column.发展新柱型发展新柱型nHelp us to choose the operating conditions of chromatography 选择色谱条件选择色谱条件nHelp us to prepare a high efficient column.制备高效柱制备高效柱The other factors result in the peak width broadening 影响色谱峰展宽的其他因素影响色谱峰展宽的其他因素nNonlinear pa

16、rtition chromatography 非线性分配非线性分配nTailing peak caused by the active points on the stationary phase.固定相的活性吸附点固定相的活性吸附点nExtra-column effects:柱外效应柱外效应nThe factors result in the broadening out of the column,Such as the injection system,dead volume of detector et al.4.色谱条件优化理论色谱条件优化理论分离度的定义nA good chroma

17、tographic separation is judged by resolution between peeks.R用以评判色谱分离情况用以评判色谱分离情况nThis definition concludes dynamic and thermodynamic factors.It can evaluate the total separation efficiency of column.R是评价柱分离总效能的指标是评价柱分离总效能的指标Resolution Equation 分离度（分离度（R）方程）方程提高提高n,k均可提高均可提高分离度分离度为色谱条件的优化提供理论指导为色谱条件的

18、优化提供理论指导5.Qualitative chromatographic analysis and quantitative chromatographic analysis色谱定性与定量分析5.1 Qualitative chromatographic analysis 色谱定性分析色谱定性分析lRetention values for qualitative analysis 利用保留值定性利用保留值定性 The major qualitative analysis parameter of chromatography is retention time,because it is c

19、onstant when the conditions of chromatography is constant.nComparing with the value of standard 纯物对照纯物对照nComparing with data in documents 文献值对照文献值对照nCoupled with other methods to identify 与其它方法联合定性与其它方法联合定性nOff-line 离线方式离线方式nOn-line 在线方式在线方式GC-MS、GC-FTIR、LC-MS5.2 Quatitative chromatographic analysis

20、 色谱定量分析色谱定量分析nQuatitative chromatographic analysis is based on the following equation:Quantitative method 三种常用定量方法三种常用定量方法nNormalising peak areas 归一化，校正归一化归一化，校正归一化nInternal standard 内标法内标法nExternal standard 外标法外标法 Normalising peak areas 峰面积归一化 corrected normalising peak areasApplication range：All c

21、omponents can be elute,all components can be detected and all components can be separated.所所有有组组分分都都能能出出峰、检测并分离峰、检测并分离Advantage：simple and accurate，chromatographic conditions has few effect on the result.It is suitable for the quantitation of high concentration samples.简单、准确，条件简单、准确，条件/进样量对结果影响不大进样量

22、对结果影响不大Disadvantage：the usage is limited.使用受限使用受限 internal standard 内标法内标法Application range：for accurate quantitation。精确定量精确定量advantage：chromatographic conditions has few effect on the result.No limits for separation and detection。色色谱谱条条件件，进进样样量对结果影响小量对结果影响小disadvantage：operation relative complex,an

23、alysis time will be prolonged.操作相对复杂些操作相对复杂些Regular analysis 常规分析常规分析advantage：simple,convenient 简单方便简单方便disadvantage：the accurate of result is determined by reproducibility of injection and stability of chromatographic conditions.结结果果的的准准确确性性取取决决于于进进样样的的重重复复性性和和色色谱谱条条件件的稳定性的稳定性 External standard(working curve)外标法（工作曲线法）外标法（工作曲线法）5.2.4 The error in quantitation 定量误差定量误差nRepresentation of samples 样品的代表性样品的代表性nThe error caused by injector 进样误差进样误差nThe error caused by column 色谱柱引起的误差色谱柱引起的误差nMeasurement error of peak areas 峰面积测量峰面积测量nMeasurement uncertainty 不确定度不确定度