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1、Chapter Six High Performance Liquid ChromatographyHigh Performance Liquid Chromatography（HPLCHPLC）第六章 高效液相色譜法高效液相色譜法高效液相色譜法 HPLCHigh Performance Liquid Chromatography液相色譜流程圖液相色譜流程圖PUMPDetectorWData station (Recorder) or Collection王秋泉王秋泉 博士博士廈門大學(xué)化學(xué)系，教育部現(xiàn)代分析科學(xué)重點(diǎn)實(shí)驗(yàn)室廈門大學(xué)化學(xué)系，教育部現(xiàn)代分析科學(xué)重點(diǎn)實(shí)驗(yàn)室Tel: 2181796; E

2、mail: 2001-2004高效液相色譜法高效液相色譜法 HPLCHigh Performance Liquid Chromatography往復(fù)式高壓恒流泵高效液相色譜法高效液相色譜法 HPLCHigh Performance Liquid Chromatography注射泵高效液相色譜法高效液相色譜法 HPLCHigh Performance Liquid Chromatography梯度洗脫（淋洗）低壓梯度高壓梯度PUMPPUMPPUMP濃度時(shí)間高效液相色譜法高效液相色譜法 HPLCHigh Performance Liquid Chromatography液相色譜流程圖液相色譜流程圖

3、PUMPDetectorWData station (Recorder) or Collection正相色譜正相色譜 反相色譜反相色譜 Reverse-phase ChromatographyReverse-phase Chromatography液固色譜液固色譜Liquid-Solid Chromatography液固吸附色譜液固吸附色譜Liquid-Solid ChromatographymadadmnMXnMX+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液液分配色譜Liquid-Liquid ChromatographyaqorgiiKiSt

4、ationary phaseMobile phase固定液易流失 化學(xué)鍵合固定相化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase ChromatographySiO H+ ROHSiOR + H2OSiO H+ R3SiClSiOSiR3 + H C l1 功能基團(tuán)的引入方法功能基團(tuán)的引入方法SO C l2C lR MgC lRSiOHSiClSOCl2RM gClSiR化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase Chromatography化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase ChromatographyR-C4, -C8, -C18-C6H5, -CN, -N

5、H2-SO3H, -COOH, -NR4Cl-化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase Chromatography2正相色譜正相色譜3 反相色譜反相色譜 RP-HPLCRP-HPLC4 反相離子對(duì)色譜反相離子對(duì)色譜 RP-Ion-Pair HPLCRP-Ion-Pair HPLCNSO3HONSO3HOM2+NSO3HOHNM(HQS)n2-n反相離子對(duì)色譜反相離子對(duì)色譜 RP-Ion-Pair HPLCRP-Ion-Pair HPLCMinutes02468101214Volts-0.10.00.10.20.30.4Volts-0.10.00.10.20.30.4 MgCdZn

6、Chromatogram of Mg, Cd and Zn separation化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase Chromatography5 離子交換色譜離子交換色譜COOHCOO-COO-COOHCOOHCOOHCOOHCOOHCOO-COO-COO-COO-COOHCOO-Figure Interaction between REE-species and the carboxylic groupsof the ion exchangerREE3+REE3+nHmLj-REE(HmL)n(3-nj)+Mobile phase flownMinutes05101520

7、2530mAU0246mAU02461 1.088 29.872 11.048 44.48 3 18.948 25.661: 658 nm, 8 nm430Soil-D20-3000Pk #Retention TimeArea Percent化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase ChromatographyDistribution of different REEs species inthe soluble extract of the soil sample29. 87%44. 48%25. 66%化學(xué)鍵合相色譜化學(xué)鍵合相色譜Bonded Phase Chromatogra

8、phy空間排斥（阻）色譜空間排斥（阻）色譜Size Exclusion Chromatography硅膠型、聚苯乙烯型、葡聚糖凝膠型硅膠型、聚苯乙烯型、葡聚糖凝膠型空間排斥（阻）色譜空間排斥（阻）色譜Size Exclusion ChromatographyM.W.tR空間排斥（阻）色譜空間排斥（阻）色譜Size Exclusion ChromatographyM.W.tR空間排斥（阻）色譜空間排斥（阻）色譜Size Exclusion ChromatographyM.W.tR空間排斥（阻）色譜空間排斥（阻）色譜Size Exclusion ChromatographyM.W.tR空間排斥（阻

9、）色譜空間排斥（阻）色譜Size Exclusion ChromatographyM.W.tRConditions: Column, Vydac C18, 5-mm, 30 nm (1 mm 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 minSample:

10、mixture of 9 protein standards; 5-mL loopDetection: SCIEX TAGA 6000E triple quadrupole mass spectrometry equipped with ionspray atmospheric pressure ion source in positive ion mode1, substance P(1-9),MW=1104, 900 pmoles; 2, neurotensin, 1673, 150;3, angiotensin, 1296, 385; 4, somatostatin, 1638, 600

11、; 5, bovine insulin,5732, 130; 6, bovine glucagon, 3482, 720; 7, mellitin, 2846, 175; 8, myo-Globin, 16950, 20; 9, lactoglobulin-B, 18277, 245.m m-HPLC-Mass Spectrometry -HPLC-Mass Spectrometry of Peptides and Proteinsof Peptides and Proteinsm m-HPLC-Mass Spectrometry -HPLC-Mass Spectrometry of Pept

12、ides and Proteinsof Peptides and ProteinsMass Spectrum Mass Spectrum of Substance P from Peak 1of Substance P from Peak 1Mass Spectrum Mass Spectrum of Bovine Insulin from Peak 5of Bovine Insulin from Peak 5Mass Spectrum Mass Spectrum of Bovine Glucagon of Bovine Glucagon ( (胰增血糖素）胰增血糖素）from Peak 6f

13、rom Peak 6Mixed Mass Spectrum from Peak 8Mixed Mass Spectrum from Peak 8Separation and detection of the pigments in narcissus leaves by m m-HPLC-APCI-MSSeparation and detection of the pigments in narcissus leaves by m m-HPLC-APCI-MSSeparation and detection of the pigments in narcissus leaves by m m-

14、HPLC-APCI-MSSeparation and detection of the pigments in narcissus leaves by m m-HPLC-APCI-MSSeparation and detection of the pigments in narcissus leaves by m m-HPLC-APCI-MSSeparation and detection of the pigments in narcissus leaves by m m-HPLC-APCI-MSSeparation and detection of the pigments in narcissus leaves by m m-HPLC-APCI-MS