1、,高速逆流色譜原理及應(yīng)用,多級萃取技術(shù),離心分配色譜平面圖,離心分配色譜立體圖,同步行星式逆流色譜,高速逆流色譜運行平面圖,分離區(qū),分離區(qū),混合區(qū),HSCCC 系統(tǒng)連接工作圖,1、HPLC方法的建立 盡可能使各峰得到良好的分離 2、溶劑系統(tǒng)的選擇 根據(jù)樣品的溶解特性選擇溶劑體系 測定分配系數(shù)，依據(jù)分配系數(shù)確定溶劑系統(tǒng) K2/K1:0.5-2.5 3、HSCCC分離條件優(yōu)化 色譜柱轉(zhuǎn)速；固定相的保留；流動相流速；分離溫度。 4、HPLC測定收集組分的純度,HSCCC方法的建立過程:,丁醇-醋酸-水丁醇-甲醇-水(緩沖液) 丁醇-乙酸乙酯-水(緩沖液) 乙酸乙酯-水(緩沖液),氯仿-甲醇-水(毒)

2、 甲基異丁基酮-丙酮-水 正己烷-乙酸乙酯-甲醇-水 (石油醚-乙酸乙酯-甲醇-水),正己烷-乙腈 正己烷-甲醇-水 正己烷-乙酸乙酯-甲醇-水 (石油醚-乙酸乙酯-甲醇-水),水溶性和強極性,中等極性(氯仿),弱極性(正己烷),HSCCC常用溶劑系統(tǒng)（依據(jù)組分的極性和溶解性）,Preparative Isolation of Six Major Saponins from Platycodi Radix by High-speed Counter-current Chromatography,Young Wan Ha and Yeong Shik Kim,ABSTRACT: Introduc

3、tion Platycosides, the primary constituents of Platycodi Radix, are known to have numerous and varied biological activities, exerting anti-inflammation, anti-allergy, anti-tumour, anti-obesity and anti-hyperlipidemia effects. However, effective methods for isolating and purifying platycosides from P

4、latycodi Radix are not currently available. Objective To develop an efficient method for the preparative separation of six platycosides from Platycodi Radix by high speed counter-current chromatography (HSCCC) coupled with an evaporative light scattering detection (ELSD) system. Methodology Preparat

5、ive separation was performed by water extraction using reversed-phase C18 column chromatography on an HSCCC-ELSD system. A two-phase solvent system comprised hexanen-butanolwater (1:40:20, v/v) and (1:10:5, v/v) was employed. Two other key parameters, revolution speed of the separation column and fl

6、ow-rate of the mobile phase, were also investigated for optimum HSCCC performance. Each peak fraction obtained from separation of the platycosides was collected according to the ELSD elution profile and determined by HPLC. Results Using the described method, six platycosides, all with purities of ov

7、er 94%, could be isolated from 300mg of the platycoside-enriched fraction. Their structures were characterized by electrospray ionisation mass spectrometry (ESI-MS), 1HNMR and 13C-NMR.,Introduction,Experimental,Apparatus.,Reagents and materials.,Preparation of crude sample.,Measurement of partition

8、coefficients,Preparation of the two-phase solvent system and sample solution.,HSCCC separation,Selection of the two-phase solvent system and HSCCC conditions,Results and Discussion,HPLC analysis and identification of HSCCC peak fractions,Structural identification,Acknowledgements,References,高速逆流色譜的延

9、伸應(yīng)用,實例1 pH區(qū)帶CCC分離同分異構(gòu)體,HSCCC條件： 溶劑系統(tǒng):正丁醇-水(1:1) 固 定 相:上相pH=0.5(HCl) 流 動 相:下相pH=10.7(NH3) 流 速:2.0 ml min-1 轉(zhuǎn) 速:715 rpm 檢測波長:206 nm 樣 品 量:230 mg 粗提物 機 型:pH-zone-refining CCC 固定相保留： 60% 1: 3-sulfophthalic acid 99.0 2: 4-sulfophthalic acid 99. 0,Journal of Chromatography A, 966 (2002) 111118,pH區(qū)帶逆流色譜分離中

10、藥單體中應(yīng)用,實例2 CCC分離蛇床子,HSCCC條件： 溶劑系統(tǒng): 正己烷-乙酸乙酯-乙醇-水(5:5 :5:5) 流 速: 0 180 min, 1ml min1 180480 min, 2ml min1 轉(zhuǎn) 速:1000 rpm 檢測波長:254 nm 樣 品 量:500 mg 粗提物 機 型:CCC-1000 相 保 留:58% A: bergapten 96.5% 45.8 mg B: imperatorin 98.2% 118.3 mg 92.1 、93.7%,Journal of Chromatography A, 1061 (2004) 5154,逆流色譜分離中藥應(yīng)用(V流動相

11、),實例3 CCC分離短瓣金蓮花黃酮苷,HSCCC條件： 溶劑系統(tǒng):乙酸乙酯-正丁醇-水(2:1:3) 流 速: 0 190 min, 1.5ml min1 190480 min, 2.5ml min1 轉(zhuǎn) 速:800 rpm 檢測波長:254 nm 樣 品 量:500 mg 粗提物 機 型:CCC-1000 保 留 值:49% 1: quercetin-3-O-neohesperidoside 85.1% 9.3 mg 2: orientin(葒草素) 97% 95.8 mg 3: 未知化合物 97% 4: vitexin(牡荊素) 97% 11.6 mg,Journal of Chroma

12、tography A, 1092 (2005) 216221,逆流色譜分離中藥應(yīng)用(V流動相),1.5 ml/min,2.5 ml/min,貫葉連翹中金絲桃苷,HSCCC-HPLC-DAD聯(lián)用分離中藥應(yīng)用,三相CCC分離15種化合物,逆流色譜分離中藥應(yīng)用(三相溶劑系統(tǒng)),HEXMeOAcACN-Water,實例6 二維CCC分離冬凌草,HSCCC條件： 溶劑系統(tǒng): 一維:正己烷-乙酸乙酯-甲醇-水 1:5:1:5 二維:正己烷-乙酸乙酯-甲醇-水 3:5:3:5 流 速 :2ml min1 轉(zhuǎn) 速 :800 rpm 樣 品 量 :250 mg 粗提物 機 型: 2D-CCC 保 留 值:一維:

13、 40% 二維: 63% 1: oridonin (甲素) 95% 2: ponicidin (乙素) 95%,Journal of Chromatography A, 1146 (2007) 125130,二維逆流色譜分離中藥應(yīng)用,實例7 分離白花前胡中香豆素,HSCCC條件： 溶劑系統(tǒng): 石油醚-乙酸乙酯-甲醇-水 5:5:6:4 流 速 :2ml min1 轉(zhuǎn) 速 :900 rpm 樣 品 量 :250 mg 粗提物 機 型 :TBE-300 保 留 值 :54.3% 1: 7.0 mg 99.3% 2: 7.4 mg 96.1% 3: 12.5mg 96.5% 4: 42.1mg 99

14、.3% 5: 9.8 mg 99.7% 6: 65.2 mg 98.1% 7: 31.7mg, 92.8%,Journal of Chromatography B, 877 (2009) 25712578,HSCCC-ESI-MSn分離中藥應(yīng)用,擴展和想象,湖州:活豬走進(jìn)去 鮮肉運出來 全機械化操作,中國豬業(yè)網(wǎng)2010-12-27,由于該定點屠宰遷建項目采用自動化同步流水線加工，活豬進(jìn)去，出來便是新鮮的豬肉，減少了手工的環(huán)節(jié)和過程，豬肉更衛(wèi)生，肉品質(zhì)量也會有大幅提高。,Application of accelerated solvent extraction coupled with high

15、-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L.,Yuchi Zhang, Chunming Liu, Min Yu, Zhengkun Zhang, Yanjuan Qi, Jing Wang, Guimei Wu, Sainan Li, Jing Yu, Yang Hu,Journal of Chromatography A, 1218 (2011) 28272834,Hype

16、ricum perforatum L., a typical traditional Chinese medicine, is used all over the world for the treatment of depression. Additionally, it has been reported that the extracts of H. perforatum may have an inhibitory effect, on human bladder cancer cells , and act as an antioxidant and anticonvulsant.

17、The extracts of H. perforatum contain many constituents with documented biological activity such as quinic acids, phloroglucinols and a broad range of flavonoids . Exposure of the extracts of H. perforatum to light may lead to the degradation of phloroglucinols, which are extremely sensitive to oxid

18、ation and unstable in solution on exposure to air, therefore phloroglucinols are difficult to separate and isolate by conventional method. In this case, amethod combining an extraction system with an isolation system online to avoid the exposure of the extracts to air and light is urgently needed.,I

19、ntroduction,The diagram presents two setups (B) enabling hyphenation of ASE and HPCCC,電視劇東北一家人 背景：汽車廠家屬區(qū)開辟一片荒地，打算承包給個人，由個人決定所要開發(fā)的娛樂項目。 甲（牛小偉，呂小品扮演）：打算建設(shè)過山車。 乙（牛繼紅，金珠扮演） ：打算開發(fā)滑梯和秋千相關(guān)的項目。 丙（孫明，鞏漢林扮演） ：認(rèn)為甲和乙所開發(fā)的項目太過時，小兒科，丙的思路是在 過山車上打滑梯，蕩秋千。,擴展和想象,Completed preparative separation of alkaloids from Cepha

20、ltaxus fortunine by step-pH-gradient high-speed counter-current chromatography,Zhonghua Liu, Qizhen Du, Kuiwu Wang, Lili Xiu, Guanglei Song,Journal of Chromatography A, 1216 (2009) 46634667,Chemical structure of cephalotaxine-type alkaloids,The HSCCC experimentwas performed with a procedure: The col

21、umn was first entirely filled with the organic stationary phase containing TFA. This was followed by sample injection. Then the aqueous phase containing 2% NH4OH (eluter base) was pumped into the inlet of the column at a flow-rate of 3.0 mL/min in the head to tail elution mode, while the apparatus was rotated at 750 rpm. After peaks I and II were eluted out of the column, the mobile phase was changed to the aqueous phase containing 0.2% NH4OH until peak III were eluted out. Following this, the mobile phase was changed to the aqueous phase containing 0.01% TFA to elute peaks VI and