儀器分析第五章.ppt

1、,Chapter Five High Performance Liquid Chromatography For Short : HPLC,第五章 高效液相色譜法,高效液相色譜,高效液相色譜法 HPLC High Performance Liquid Chromatography,高效液相色譜法 HPLC High Performance Liquid Chromatography,往復(fù)式高壓恒流泵,高效液相色譜法 HPLC High Performance Liquid Chromatography,注射泵,高效液相色譜法 HPLC High Performance Liquid Chrom

2、atography,梯度洗脫（淋洗）,低壓梯度 高壓梯度,濃度,時(shí)間,高效液相色譜法 HPLC High Performance Liquid Chromatography,正相色譜 反相色譜 Reverse-phase Chromatography,液固色譜 Liquid-Solid Chromatography,液固吸附色譜 Liquid-Solid Chromatography,m,ad,ad,m,nM,X,nM,X,+,+,以樣品中各組分在固定相上的吸附作用不同來進(jìn)行分離,“相似相溶”原理 異構(gòu)體的分離,液液分配色譜 Liquid-Liquid Chromatography,Ki,St

3、ationary phase,Mobile phase,固定液易流失 化學(xué)鍵合固定相,化學(xué)鍵合相色譜 Bonded Phase Chromatography,1 功能基團(tuán)的引入方法,S,O,C,l,2,C,l,R,M,g,C,l,R,S,i,O,H,S,i,C,l,S,O,C,l,2,R,M,g,C,l,S,i,R,化學(xué)鍵合相色譜 Bonded Phase Chromatography,化學(xué)鍵合相色譜 Bonded Phase Chromatography,R,-C4, -C8, -C18 -C6H5, -CN, -NH2 -SO3H, -COOH, -NR4Cl -,化學(xué)鍵合相色譜 Bond

4、ed Phase Chromatography,正相色譜 3 反相色譜 RP-HPLC 4 反相離子對色譜 RP-Ion-Pair HPLC,反相離子對色譜 RP-Ion-Pair HPLC,Chromatogram of Mg, Cd and Zn separation,化學(xué)鍵合相色譜 Bonded Phase Chromatography,5 離子交換色譜,化學(xué)鍵合相色譜 Bonded Phase Chromatography,化學(xué)鍵合相色譜 Bonded Phase Chromatography,空間排斥（阻）色譜 Size Exclusion Chromatography,硅膠型、聚苯

5、乙烯型、葡聚糖凝膠型,空間排斥（阻）色譜 Size Exclusion Chromatography,M.W.,tR,空間排斥（阻）色譜 Size Exclusion Chromatography,M.W.,tR,空間排斥（阻）色譜 Size Exclusion Chromatography,M.W.,tR,空間排斥（阻）色譜 Size Exclusion Chromatography,M.W.,tR,空間排斥（阻）色譜 Size Exclusion Chromatography,M.W.,tR,Conditions: Column, Vydac C18, 5-mm, 30 nm (1 mm

6、ID100 mm) Flow rate, 40 mL/min Mobile phase, A: 0.05% Formic Acid in Water B: 0.05% Formic Acid in Acetonitrile Linear gradient programmed from 20% B to 100% B over 25 min,hold at 100% B for 5 min Sample: mixture of 9 protein standards; 5-mL loop Detection: SCIEX TAGA 6000E triple quadrupole mass sp

7、ectrometry equipped with ionspray atmospheric pressure ion source in positive ion mode 1, substance P(1-9),MW=1104, 900 pmoles; 2, neurotensin, 1673, 150; 3, angiotensin, 1296, 385; 4, somatostatin, 1638, 600; 5, bovine insulin, 5732, 130; 6, bovine glucagon, 3482, 720; 7, mellitin, 2846, 175; 8, my

8、o- Globin, 16950, 20; 9, lactoglobulin-B, 18277, 245.,m-HPLC-Mass Spectrometry of Peptides and Proteins,m-HPLC-Mass Spectrometry of Peptides and Proteins,Mass Spectrum of Substance P from Peak 1,Mass Spectrum of Bovine Insulin from Peak 5,Mass Spectrum of Bovine Glucagon (胰增血糖素）from Peak 6,Mixed Mas

9、s Spectrum from Peak 8,Separation and detection of the pigments in narcissus leaves by m-HPLC-APCI-MS,Separation and detection of the pigments in narcissus leaves by m-HPLC-APCI-MS,Separation and detection of the pigments in narcissus leaves by m-HPLC-APCI-MS,Separation and detection of the pigments in narcissus leaves by m-HPLC-APCI-MS,Separation and detection of the pigments in narcissus leaves by m-HPLC-APCI-MS,Separation