高速逆流色谱原理及应用

1、,高速逆流色谱原理及应用,多级萃取技术,离心分配色谱平面图,离心分配色谱立体图,同步行星式逆流色谱,高速逆流色谱运行平面图,分离区,分离区,混合区,HSCCC 系统连接工作图,1、HPLC方法的建立 尽可能使各峰得到良好的分离 2、溶剂系统的选择 根据样品的溶解特性选择溶剂体系 测定分配系数，依据分配系数确定溶剂系统 K2/K1:0.5-2.5 3、HSCCC分离条件优化 色谱柱转速；固定相的保留；流动相流速；分离温度。 4、HPLC测定收集组分的纯度,HSCCC方法的建立过程:,丁醇-醋酸-水丁醇-甲醇-水(缓冲液) 丁醇-乙酸乙酯-水(缓冲液) 乙酸乙酯-水(缓冲液),氯仿-甲醇-水(毒)

2、 甲基异丁基酮-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),水溶性和强极性,中等极性(氯仿),弱极性(正己烷),HSCCC常用溶剂系统（依据组分的极性和溶解性）,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,高速逆流色谱的延

9、伸应用,实例1 pH区带CCC分离同分异构体,HSCCC条件： 溶剂系统:正丁醇-水(1:1) 固 定 相:上相pH=0.5(HCl) 流 动 相:下相pH=10.7(NH3) 流 速:2.0 ml min-1 转 速:715 rpm 检测波长:206 nm 样 品 量:230 mg 粗提物 机 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区带逆流色谱分离中

10、药单体中应用,实例2 CCC分离蛇床子,HSCCC条件： 溶剂系统: 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 转 速:1000 rpm 检测波长:254 nm 样 品 量:500 mg 粗提物 机 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,逆流色谱分离中药应用(V流动相

11、),实例3 CCC分离短瓣金莲花黄酮苷,HSCCC条件： 溶剂系统:乙酸乙酯-正丁醇-水(2:1:3) 流 速: 0 190 min, 1.5ml min1 190480 min, 2.5ml min1 转 速:800 rpm 检测波长:254 nm 样 品 量:500 mg 粗提物 机 型:CCC-1000 保 留 值:49% 1: quercetin-3-O-neohesperidoside 85.1% 9.3 mg 2: orientin(荭草素) 97% 95.8 mg 3: 未知化合物 97% 4: vitexin(牡荆素) 97% 11.6 mg,Journal of Chroma

12、tography A, 1092 (2005) 216221,逆流色谱分离中药应用(V流动相),1.5 ml/min,2.5 ml/min,贯叶连翘中金丝桃苷,HSCCC-HPLC-DAD联用分离中药应用,三相CCC分离15种化合物,逆流色谱分离中药应用(三相溶剂系统),HEXMeOAcACN-Water,实例6 二维CCC分离冬凌草,HSCCC条件： 溶剂系统: 一维:正己烷-乙酸乙酯-甲醇-水 1:5:1:5 二维:正己烷-乙酸乙酯-甲醇-水 3:5:3:5 流 速 :2ml min1 转 速 :800 rpm 样 品 量 :250 mg 粗提物 机 型: 2D-CCC 保 留 值:一维:

13、 40% 二维: 63% 1: oridonin (甲素) 95% 2: ponicidin (乙素) 95%,Journal of Chromatography A, 1146 (2007) 125130,二维逆流色谱分离中药应用,实例7 分离白花前胡中香豆素,HSCCC条件： 溶剂系统: 石油醚-乙酸乙酯-甲醇-水 5:5:6:4 流 速 :2ml min1 转 速 :900 rpm 样 品 量 :250 mg 粗提物 机 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分离中药应用,扩展和想象,湖州:活猪走进去 鲜肉运出来 全机械化操作,中国猪业网2010-12-27,由于该定点屠宰迁建项目采用自动化同步流水线加工，活猪进去，出来便是新鲜的猪肉，减少了手工的环节和过程，猪肉更卫生，肉品质量也会有大幅提高。,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,电视剧东北一家人 背景：汽车厂家属区开辟一片荒地，打算承包给个人，由个人决定所要开发的娱乐项目。 甲（牛小伟，吕小品扮演）：打算建设过山车。 乙（牛继红，金珠扮演） ：打算开发滑梯和秋千相关的项目。 丙（孙明，巩汉林扮演） ：认为甲和乙所开发的项目太过时，小儿科，丙的思路是在 过山车上打滑梯，荡秋千。,扩展和想象,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