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1、第8章 高效液相色谱法 HPLCHigh Performance Liquid Chromatography特点：高压、高效、高速高沸点、热不稳定有机及生化试样的高效分离分析方法。Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-
2、2011 Aspose Pty Ltd.8.1 高效液相色谱仪四大部分组成：溶剂输送系统（贮液器，高压泵）进样系统（进样阀等）分离系统（色谱柱等）检测记录系统（检测器，记录仪等） Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-
3、2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspo
4、se.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slid
5、es for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.1. 高压输液泵主要部件之一，压力：150350105 Pa为了获得高柱效而使用粒度很小的固定相（Rb+K+NH4+Na+H+Li+二价阳离子：Ba2+Pb2+Sr2+Ca2+Cd2+Cu2+Zn2+Mg2+强碱性阴离子交换树脂,保留值大小顺序:PO43-SO42-C2O42-I-HSO4-NO3-CN-NO2-Cl- HCOO-OH-F-CH 3COO-Evaluation
6、only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client P
7、rofile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd. 流动相H2O、H2O + 乙醇、H2O + pH 缓冲剂 应用无机离子,有机和生物酸碱。如氨基酸、核酸、蛋白质等 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0
8、.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.4. 尺寸排阻色谱法size exclusion chromatography SEC凝胶过滤色谱: 水为流动相gel filtration chromatgraphy GFC凝胶渗透色谱: 非水流动相gel permeation chromatography GPC 1959年 Porath和Flodin发明分离原理: 试
9、样分子的尺寸和形状不同来实现分离Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.S
10、lides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides f
11、or .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.GPC流动相: 水、四氢呋喃 应 用: 聚合物的分级级生物聚合体（蛋白质质，核酸，低聚糖,多肽肽）的分离Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client
12、 Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.8.3 色谱分离方法的选择l了解样品的有关性质样品的相对分子质量的大小在水中和有机溶剂中的溶解度、极性和稳定程度化学结构l 熟悉各种色谱方法的主要特点及其应用范围Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET
13、 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyri
14、ght 2004-2011 Aspose Pty Ltd.1. 流动相种类与极性液相色谱的流动相又称为：淋洗液，洗脱剂它可显著改变组分分离状况选择流动相在液相色谱中特别重要 按组成可分为： 单组分和多组分；按极性可分为： 极性、弱极性、非极性；按使用方式可分为：固定组成淋洗和梯度淋洗常用溶剂:己烷、四氯化碳、甲苯、乙酸乙酯、乙醇、乙腈、水采用二元或多元组合溶剂作为流动相可以灵活调节流动相的极性或增加选择性，以改进分离或调整出峰时间Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Pro
15、file 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.选择流动相时应注意的几个问题：（1）尽量使用高纯度试剂作流动相，防止微量杂质长期累 积损坏色谱柱和使检测器噪声增加。（2）避免流动相与固定相发生作用而使柱效下降或损坏 柱子。如使固定液溶解流失；酸性溶剂破坏氧化硅固定 相等。（3）试样在流动相中应有适宜的溶解度，防止产生沉 淀并在柱中沉积。（4）流动相同时
16、还应满足检测器的要求。当使用紫外 检测器时，流动相不应有紫外吸收。Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.8.4 应用实例方针：要解决一个分析问题，先试反相色谱法C18标准柱，改变流动相调节k（保留行为）和（选择性）反相色谱法优点：a.色谱柱平衡快，适宜梯度洗脱b.流动相主体为水，甲醇为有机改性剂，价廉易纯化c.tR 随溶质的疏水性增加而增大，可预计出峰顺序d.改变基本流动相的pH，添加盐缓冲剂，金属螯和物，
