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1、张语迟张语迟高速逆流色谱原理、应用高速逆流色谱原理、应用及带给我们的启发思考及带给我们的启发思考 20132013年年4 4月月1 1日日色谱的来源色谱的来源气象色谱气象色谱逆流色谱逆流色谱液相色谱液相色谱介质为气体介质为液体介质为固体气象色谱气象色谱逆流色谱逆流色谱液相色谱液相色谱介质为气体介质为液体介质为固体固相萃取固相萃取介质为固体大雁在空气中飞行大雁在空气中飞行大雁在迁徙过程要飞行大雁在迁徙过程要飞行3000-40003000-4000公里公里气象色谱柱长度为气象色谱柱长度为3030米，各类色米，各类色谱柱长度相比，气相色谱柱最长谱柱长度相比，气相色谱柱最长西伯利亚西伯利亚中国南方中国
2、南方气象色谱气象色谱介质为气体鱼类在水中游动鱼类在水中游动鱼类游动距离一般为鱼类游动距离一般为500-1000500-1000公里公里逆流色谱柱长度为逆流色谱柱长度为20-3020-30米，介于米，介于气相色谱柱和液相色谱柱中间气相色谱柱和液相色谱柱中间逆流色谱逆流色谱介质为液体蚯蚓在泥土中慢慢蠕动蚯蚓在泥土中慢慢蠕动蚯蚓蠕动距离一般不超过蚯蚓蠕动距离一般不超过5050米米液相色谱柱长度一般不超过液相色谱柱长度一般不超过2525厘米，厘米，在常见色谱柱中长度最短在常见色谱柱中长度最短院东院东院西院西液相色谱液相色谱介质为固体多级萃取技术多级萃取技术离离心心分分配配色色谱谱平平面面图图HPCCC
3、离心分配色谱立体图离心分配色谱立体图HPCCC同步行星式逆流色谱同步行星式逆流色谱HSCCCHSCCC高速逆流色谱运行平面图HSCCCHSCCC分离区分离区分离区分离区混合区混合区流体静力学平衡流体静力学平衡(HSES)流体动力学平衡流体动力学平衡(HDES)逆流色谱的基本体系示意图逆流色谱的基本体系示意图高速逆流色谱高速逆流色谱(HSCCC)(HSCCC)的原理的原理HSCCC 系统连接工作图HPCPCHSCCCHPCCCHPCCC和和HSCCCHSCCC对比对比优点：固定相和流动相充分优点：固定相和流动相充分混合，分离速度快。混合，分离速度快。缺点：对含有正丁醇等黏性缺点：对含有正丁醇等黏
4、性较大的溶剂系统保留不好。较大的溶剂系统保留不好。优点：对含有正丁醇等黏性优点：对含有正丁醇等黏性较大的溶剂系统保留好。尤较大的溶剂系统保留好。尤其适合皂苷类成分的分离其适合皂苷类成分的分离优点：固定相和流动相混合优点：固定相和流动相混合速度较慢，分离速度慢。速度较慢，分离速度慢。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/K1:0.5-2.53、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HSCCCHSCCC方法的建立过程: :正丁醇-甲醇-
5、水(缓冲液)正丁醇-乙酸乙酯-水(缓冲液)乙酸乙酯-水(缓冲液)氯仿-甲醇-水(毒)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 ChromatographyYoung Wan Ha and Yeon
6、g Shik KimABSTRACT:Introduction 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 pu
7、rifying platycosides from Platycodi 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) sy
8、stem.Methodology Preparative 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
9、 separation column and flow-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,
10、 all with purities of over 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.IntroductionExperimentalApparatus.Reagents and materials.Preparation of crude sample.Measure
11、ment of partition coefficientsPreparation of the two-phase solvent system and sample solution.HSCCC separationSelection of the two-phase solvent system and HSCCC conditionsResults and DiscussionHPLC analysis and identification of HSCCC peak fractionsStructural identificationAcknowledgementsReference
12、s高速逆流色谱的延伸应用高速逆流色谱的延伸应用实例实例2 CCC2 CCC分离蛇床子分离蛇床子HSCCC条件：条件：溶剂系统溶剂系统:正己烷正己烷-乙酸乙酯乙酸乙酯-乙醇乙醇-水水(5:5 :5:5)流流 速速: 0 180 min, 1ml min1 180480 min, 2ml min1转转 速速:1000 rpm检测波长检测波长:254 nm样样 品品 量量:500 mg 粗提物粗提物机机 型型:CCC-1000相相 保保 留留:58%A: bergapten 96.5% 45.8 mg B: imperatorin 98.2% 118.3 mg 92.1 、93.7% Journal
13、 of Chromatography A, 1061 (2004) 5154逆流色谱分离中药应用(V流动相)实例实例1 pH1 pH区带区带CCCCCC分离同分异构体分离同分异构体HSCCC条件：条件：溶剂系统溶剂系统:正丁醇正丁醇-水水(1:1)固固 定定 相相:上相上相pH=0.5(HCl)流流 动动 相相:下相下相pH=10.7(NH3)流流 速速:2.0 ml min-1转转 速速:715 rpm检测波长检测波长:206 nm样样 品品 量量:230 mg 粗提物粗提物机机 型型:pH-zone-refining CCC固定相保留：固定相保留： 60%1: 3-sulfophthali
14、c acid 99.02: 4-sulfophthalic acid 99. 0Journal of Chromatography A, 966 (2002) 111118pH区带逆流色谱分离中药单体中应用实例实例3 CCC3 CCC分离短瓣金莲花黄酮苷分离短瓣金莲花黄酮苷HSCCC条件：条件：溶剂系统溶剂系统:乙酸乙酯乙酸乙酯-正丁醇正丁醇-水水(2:1:3)流流 速速: 0 190 min, 1.5ml min1 190480 min, 2.5ml min1转转 速速:800 rpm检测波长检测波长:254 nm样样 品品 量量:500 mg 粗提物粗提物机机 型型:CCC-1000保保
15、留留 值值:49%1: quercetin-3-O-neohesperidoside 85.1% 9.3 mg2: orientin(荭草素荭草素) 97% 95.8 mg 3: 未知化合物未知化合物 97% 4: vitexin(牡荆素牡荆素) 97% 11.6 mg Journal of Chromatography A, 1092 (2005) 216221逆流色谱分离中药应用(V流动相)1.5 ml/min2.5 ml/min实例实例4:4:三相三相CCCCCC分离分离1515种化合物种化合物逆流色谱分离中药应用(三相溶剂系统)HEXMeOAcACN-Water实例实例5 5 二维二维
16、CCCCCC分离冬凌草分离冬凌草HSCCC条件：条件：溶剂系统溶剂系统:一维一维:正己烷正己烷-乙酸乙酯乙酸乙酯-甲醇甲醇-水水 1:5:1:5二维二维:正己烷正己烷-乙酸乙酯乙酸乙酯-甲醇甲醇-水水 3:5:3:5流流 速速 :2ml min1 转转 速速 :800 rpm样样 品品 量量 :250 mg 粗提物粗提物保保 留留 值值:一维一维: 40% 二维二维: 63% 1: oridonin (甲素甲素) 95% 2: ponicidin (乙素乙素) 95%Journal of Chromatography A, 1146 (2007) 125130二维逆流色谱分离中药应用实例实例6
17、 6 分离白花前胡中香豆素分离白花前胡中香豆素HSCCC条件：条件：溶剂系统溶剂系统:石油醚石油醚-乙酸乙酯乙酸乙酯-甲醇甲醇-水水 5:5:6:4流流 速速 :2ml min1 转转 速速 :900 rpm样样 品品 量量 :250 mg 粗提物粗提物机机 型型 :TBE-300保保 留留 值值 :54.3% 1: 7.0 mg 99.3% 2: 7.4 mg 96.1% 3: 12.5mg 96.5% 4: 42.1mg 99.3% 5: 9.8 mg 99.7% 6: 65.2 mg 98.1% 7: 31.7mg, 92.8%Journal of Chromatography B, 8
18、77 (2009) 25712578HSCCC-ESI-MSn分离中药应用扩展和想象湖州湖州: :活猪走进去活猪走进去 鲜肉运出来鲜肉运出来 全机械化操作全机械化操作 中国猪业网中国猪业网2010-12-27 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，节省了大量人力物力。同时，猪肉更卫生，肉品质量也会有大幅提高。 Application of accelerated solvent extraction coupled with high-performance counter-current chromatog
19、raphy 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 HuJournal of Chromatography A, 1218 (2011) 28272834Hypericum perforatum L., a typical traditiona
20、l 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. The extracts of H. perforatum contain man
21、y 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 oxidation and unstable in solution on exposur
22、e 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.IntroductionThe diagram presents two setups
23、 (B) enabling hyphenation of ASE and HPCCC电视剧电视剧东北一家人东北一家人背景：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。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。甲（牛小伟，吕小品扮演）：打算建设过山车。乙（牛继红，金珠扮演）乙（牛继红，金珠扮演） ：打算开发滑梯和秋千相关的项目。：打算开发滑梯和秋千相关的项目。丙（孙明，巩汉林扮演）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：认为甲和乙所开发的项目太过时，小儿
24、科，丙的思路是在 过山车上打滑梯，荡秋千。过山车上打滑梯，荡秋千。扩展和想象18:15冲击力两种动态娱乐同时进行秋千转盘Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatographyZheng Zhenjia, Wang Minglin, Wang Daijie, et al.Journal of Chromatography B, 878 (2010) 16471651the new quat
25、ernary biphasic solvent systems composed of petroleum etherethyl acetatemethanolwater at volume ratios of 5:5:5:5 and 5:5:2:8 were tested. After trying both of the above solvent systems, we found the solvent system petroleum etherethyl acetatemethanolwater 5:5:2:8 (v/v/v/v) where triethylamine (10mM
26、) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5mM) to the aqueous mobile phase as an eluent was suitable for the preparative separation.petroleum etherethyl acetatemethanolwater (5:5:2:8, v/v/v/v), 10mM TEA in the upper organic stationary phase and 5mM HCl in
27、 the lower phaseCompleted preparative separation of alkaloids from Cephaltaxus fortunine by step-pH-gradient high-speed counter-current chromatographyZhonghua Liu, Qizhen Du, Kuiwu Wang, et al.Journal of Chromatography A, 1216 (2009) 46634667The column was first entirely filled with the organic stat
28、ionary 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 elu
29、ted 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 VII.mobile phase: 2% NH3, 0.2% NH3 and 0.01% TFAIn pH-zone-refin
30、ing HSCCC, an almost equal amount and less than 20mM of the acid and base are used as retainer and eluter.When an acid is used as retainer, the pH value of eluent containing target compounds is less than 7. The present separation, the content of NH4OH was much more than the content of TFA in station
31、ary phase, which lead to a pH value higher than 8 at the front of eluent. Moreover the peaks were not concentrated rectangular peaks like those of pH-zone-refining HSCCC. Therefore, the isolation should depend on the change of partition coefficients of analytes at different pH conditions.Why not pH-zone-refining HSCCC其它创意请同学其它创意请同学继续探索研究继续探索研究化学成分被吸附在固定相中，为见光明，逆流而上，从而洗脱出来。逆流色谱思考总结黄河中金背鲤鱼本来生活惬意，为了实现价值，逆流而上，开拓进取。启启 发发 和和 思思 考考鲤鱼逆流而上，实现自己的理想鲤鱼逆流而上，实现自己的理想鲤鲤鱼鱼跳跳龙龙门门黄河三尺鲤，本在孟津居。点额不成龙，归来伴凡鱼。李白祝愿大家学习进步，生活开心祝愿长师更名成功，越办越好！毛泽东
