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7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Program

January, August 6, 2012

8:30 – 9:00 Registration

9:00 – 9:10 Opening CCC 2012 Chairman: Prof. Qizhen Du

9:10 – 9:20 Welcome speech from the director of Zhejiang Gongshang University

Session 1 – CCC Keynotes Chirman: Prof. Guoan Luo

pH-zone-refining countercurrent chromatography : USA 09:20-09:50 Ito, Y. Origin, mechanism, procedure and applications K-1

Sutherland, I.*; Hewitson. P.; Scalable technology for the extraction of UK 09:50-10:20 Janaway, L.; Wood, P; pharmaceuticals (STEP): Outcomes from a year Ignatova, S. collaborative researchprogramme K-2

10:20-11:00 Tea Break with Poster & Exhibition session 1

France 11:00-11:30 Berthod, A. Terminology for countercurrent chromatography K-3

API recovery from pharmaceutical waste streams by high performance countercurrent UK 11:30-12:00 Ignatova, S.*; Sutherland, I. chromatography and intermittent countercurrent K-4 extraction

12:00-13:30 Lunch break

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 January, August 6, 2012

Session 2 – CCC Instrumentation I Chirman: Prof. Ian Sutherland

Pro, S.; Burdick, T.; Pro, L.; Friedl, W.; Novak, N.; Qiu, A new generation of countercurrent separation USA 13:30-14:00 F.; McAlpine, J.B., J. Brent technology O-1 Friesen, J.B.; Pauli, G.F.*

Berthod, A.*; Faure, K.; A small volume hydrostatic CCC column for France 14:00-14:20 Meucci, J.; Mekaoui, N. full and quick solvent selection O-2

Construction of a HSCCC apparatus with Du, Q.B.; Jiang, H.; Yin, J.; column capacity of 12 or 15 liters and its China Xu, Y.; Du, W.; Li, B.; Du, application as flash countercurrent 14:20-14:40 O-3 Q.* chromatography in quick preparation of (-)-epicatechin

14:40-15:30 Tea Break with Poster & Exhibition session 2

Session 3 – CCC Instrumentation II Chirman: Prof. Guido Pauli

Steady-state and non-steady state operation of Russia 15:30-15:50 Kostanyan, A. E. countercurrent chromatography devices O-4

True moving bed CPC for continuous France 15:50-16:10 G. Audo*; Le Quémeneur, C. purification of natural substances O-5

Goll, J.; Frey, A.; Minceva, Design of a continuous centrifugal partition Germany 16:10-16:30 M.* chromatography O-6

Hewitson, P.*; Sutherland, I.; Intermittent countercurrent extraction: Scale up UK 16:30-16:50 Wood, P.; Ignatova, S. and an improved bobbin design O-7

18:00-19:30 Welcome dinner at a restaurant

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 February, August 7, 2012

Session 4 – CCC Theory I Chirman: Prof. Alain Berthod

Selection of the mobile and stationary phase in Germany 09:00-09:20 Frey, A.*; Minceva, M. support free liquid-liquid chromatography applying a predicative thermodynamic model O-8

Schwienheer, C.*; Selection and use of aqueous two phase systems Germany 09:20-09:40 Adelmann,S.; Merz, J.; in centrifugal partition chromatography Schembecker, G. O-9

Modeling counter-current chromatography using China 09:40-10:00 Wei, Y.*; Wang, F.; Wang, S. a temperature dependence plate model O-10

China, Wu, S. ; Wu, D. ; Liang, J. ; Modeling gradient elution in CCC: Efficient 10:00-10:20 France Berthod, A.* separation of tanshinones O-11

12:40-11:20 Tea Break with Poster & Exhibition session 3

Session 5 – CCC Theory II Chirman: Prof.: Peter Winterhalter

Merz, J.*; Schwienheer, C.; Investigation of hydrodynamics in CPC Germany 11:00-11:20 Adelmann, S.; Schembecker, chambers using image processing and G. computational fluid dynamics O-12

Research of interaction between ionic and tea Chen, X.; Huang, X.; Wang, polyphenols by computer simulation assisted China 11:20-11:40 G.; Zhang J.; Di, D. * high-performance counter-current O-13 chromatography

Modeling of step wise gradients in HSCCC; A Shehzad, O.; Khan, S.; Ha, rapid and economical strategy for one step Korea 11:40-12:00 I.J.; Lee, K.J.; Tosun, A.; separation of eight dihydropyranocoumarins Kim, Y.S.* O-14 from Seseli resinousm.

12:00-13:30 Lunch break

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 February, August 7, 2012

Session 6 – CCC Application I (Room A) Chirman: Prof. Xueli Cao

Winterhalter, P.*; Jerz, G.; Isolation of bioactive polyphenols from Germany 13:30-13:50 Kuhnert, S.; Juadjur, A.; by-products of food processing using Macke,S. countercurrent chromatography O-15

Chen, X.; Huang, X.; Wang, Study on preparative separation and purification China 13:50-14:10 X.; Shi, M.; Wang, G.; of chemical compounds from plants by HSCCC Zhang, J.; Di, D.* O-16

Enantioseparation of 2-phenylpropionic acid by Zheng, Y.; Tong, S. Q.*; Yan, recycling high-speed countercurrent China 14:10-14:30 J. Z. chromatography using hydroxypropyl-β- O-17 cyclodextrin as chiral selector

Excellent fraction effect of HSCCC on the Jin,Y. Y.; Qian, D. Y.; Du, Q. China 14:30-14:50 completed separation of amide compounds in Z.* black pepper O-18

Session 7 – CCC Application II (Room B) Chirman: Prof. Eiichi Kitazume

Multifunctional tetranor triterpenoids from Hummel, H. E.*; Hein, D. F.; Azadirachta indica : A separation challenge Germany 13:30-13:50 Ma, Y.; Ito, Y. solved by MLCCC at both the analytical and O-19 preparative scale

Bioassay guided separation of bioactive components in natural products via China 13:50-14:10 Hu, R.; Chen, P.; Pan, Y.* countercurrent chromatography coupled with O-20 LC/MS/MS

He, Z. C.; Ye, Q. X.; Wu, Separation and purification of quanternary J.Y.; Yang, D. P.; Brown, L.; China 14:10-14:30 protoberberine alkaloids from Corydalis Jiang, L.; Zhu, L. P.; Wang, saxicola Bunting by HSCCC O-21 D. M.*

Separation and purification of four lignans from Yang, M.; Xu, X.*; Xie, Z.; China 14:30-14:50 Fructus arctii by high-speed countercurrent Huang, J.; Xie, C. chromatography O-22

14:50-15:30 Tea Break with Poster & Exhibition session 4

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 February, August 7, 2012

Session 8 – CCC Application III (Room A) Chirman: Prof. Hans E. Hummel

Separation andenrichment of rare earth elements Shibukawa, M.*; Shimizu, by stepwise pH gradient countercurrent Japan 15:30-15:50 K.; Saito, S. chromatography with a polyethylene O-23 glycol-Na2SO4 aqueous two-phase system

Application of high-speed countercurrent Ma, J.; Niu, L.; Cai, X.; Ito, China 15:50-16:10 chromatography, TLC and LC-MS/MS in Y.; Yang, F. lipidomics O-24

Isolation of two new diterpenoids from Xu, M.; Du, Q.;* Shen, L.; Clerodendrum kaichianum hsu. by high-speed China 16:10-16:30 Wang, H. counter-current chromatography using stepwise O-25 elution

Wang, K. B.; Liu, F.; Liu, Preparative isolation of Oolong tea polyphenols by China 16:30-16:50 Z.L.*, Huang, J. A.; Lin, high-speed countercurrent chromatography Y.; Gong, Y. S. O-26

Session 9 – CCC Application IV(Room B) Chirman: Prof. Artak Kostanyan

Ramos, R. E.; Pauli, G. F.; Generation of knock-out extracts by USA 15:30-15:50 Chen, S. N.* countercurrent chemical subtraction O-27

Rapid separation of polyphenols from green tea by Wang, W.*; Le, S.; Zhao, X; China 15:50-16:10 HSCCC and characterization the interaction Wang, T.; Zhang, J. between EGCG and protein by ACE O-28

Online isolation and purification of four phthalide compounds from Rhizome chuanxiong using China 16:10-16:30 Wei, Y.*; Huang, W. countercurrent chromatography coupled with semipreparative high performance liquid O-29 chromatography

He, M.; Du, W.; Du, Q.B.; Separation of 11-cis-retinal from the retinal China 16:30-16:50 Zhang, Y.; Li, B.; Ke, C. ; isomers by flash countercurrent chromatography Du, Q.* O-30

18:00-19:30 Gala dinner at a restaurant

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Wendesday, August 8, 2012

Session 10 – CCC Application V (Room A) Chirman: Prof. John B. Friesen

Qiu, F.; Samuel Pro, NMR-guided countercurrent purification and USA 09:00-09:20 S. ; Pauli, G. F.; Friesen, J. O-31 B.* quantification of green tea catechins O-31

Yin, L.; Cao, X. L.*; Xu, J.; Separation of bioactive components from China 09:20-09:40 Gynura divaricata (L.) DC by high-speed O-32 Cheng, C.; Pei, H. R. countercurrent chromatography O-32 Combination of high-speed countercurrent Wang, D.J., Du, J. H.*; Lin, Chromatography and Preparative HPLC to China 09:40-10:00 Separation of Flavonoid Glycosides and O-33 Y. L.; Li, S. B.; Wang, X. Caffeoylquinic Acid Derivatives from Leaves of O-33 Lonicera japonica Thunb. Preparative separation of conjugated linoleic 10:00-10:20 Song, G.L.; Wang, J.B.; acids (CLA) from camellia oleifera abel using China 10:00-10:20 Wang, Y.B.; Du, Q.Z.* pH-zone-refining counter-current O-34 chromatography Session 11 – CCC Application VI (Room B) Prof. Yuanjiang Pan

From medicinal plant to compound: isolation of Hu, P.; Liu, M.; Zhang, M.;* shikimic acid from Illicium verum by China 09:00-09:20 Hua, Y.; Zhang, H.; Wang, hyphenated expanded bed adsorption O-35 Y.; Luo, G.* chromatography and counter-current O-35 chromatography

Comprehensive separation of lignans from Lu, Y. B.*; Lin, X. J.; Wang, Zanthoxylum planispinum by countercurrent China 09:20-09:40 chromatography combined with O-36 K.W. high-performance liquid chromatography/ O-36 tandem mass spectrometry

Wang , X.; Dong, H. J.; Large-scale separation of alkaloids from China 09:40-10:00 Yang, B.; Lin, X. J.; Huang, corydalis bungeana turca.by pH-zone-refining O-37 L. Q.* counter-current chromatography O-37

Zhu, Y.; Zhan, Y.; Gui, H.; Separation of the synthetic products of China 10:00-10:20 acetylated rhein by high-speed counter-current O-38 Wei, Q.; Liu, Y.; Xu, T.* chromatography O-38 10:20-11:20 Tea Break with Poster & Exhibition session 5 Closing remarks, Edward Chou and Crafty Chromatographer Award 11:20-11:40 ceremony Prof. Qizhen Du 12:00-13:30 Lunch break 13:30-15:00 Visiting Tea Museum of China (Bus service) 15:00-16:30 Visiting Silk Museum of China (Bus service) 16:30- Tour around West Lake and Hangzhou city (transportation self service)

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 K-1 pH-zone-refining countercurrent chromatography : Origin, mechanism, procedure and applications Yoichiro Ito Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA *Correspondence address: Fax: 301-402-0013; E-mail: [email protected]

Keywords: pH-zone-refining counter-current chromatography: high-speed counter-current chromatography, preparative separation, chiral separation

This unique preparative counter-current chromatographic technology (CCC) has been developed by an incidental finding that bromoacetyl T3 formed an unusually sharp peak during its purification from a synthetic mixture. The cause of this sharp peak formation was found to be the presence of bromoacetic acid in the sample solution, which formed a sharp trailing border in the column due to its non-linear isotherm. The bromoacetyl T3 was trapped by this sharp acid border and co-eluted as a sharp peak [1]. This pH-peak-sharpening method was successfully applied for separation of a small amount of DNP-amino acids by adding three organic acids (as spacer) in the sample solution. When the sample size of each component was increased from 6 mg to 600 mg, however, each peak became a rectangular shape with a specific pH (pH zone) which, without the spacer acids in the sample solution, joined to form a single rectangular peak while preserving its sharp border, zone pH, and high purity. Hence, the method was named pH-zone-refining CCC [2]. As described above, the key element in this technology is the formation of sharp trailing border of retainer (acid or base) which moves through the column at a rate determined by its partition coefficient in the two-phase solvent system. In order to generalize the application of the method, a new way to form the sharp border between acid and base in the column was developed as follows [3]: 1) Select the two-phase solvent system which gives partition coefficient value (K) of the analyte being close to one. After equilibration in the separatory funnel, two phases are separated each in a glass container. 2) Then the retainer (organic acid such as trifluoroacetic acid for acidic analytes and organic base such as triethylamine for the basic analytes) is added to the upper organic phase while the eluter (inorganic base such as NH3 for the acidic analytes and inorganic acid such as HCl for the basic analytes) is added to the lower aqueous phase. 3) The column is first entirely filled with the upper stationary phase followed by sample injection (without pre-quilibration of the column with the aqueous phase). Then the lower aqueous mobile phase is eluted through the column. Under this experimental condition the eluter in the mobile phase gradually neutralizes the retainer present in the stationary phase to form a sharp retainer trailing border which moves through the column at a rate largely determined by the molar ratio between the retainer and the eluter. For example,10 mM retainer and 10 mM eluter will form a sharp border which moves through the column at a rate close to that of the compound with K = 1, regardless of the composition of the two-phase solvent system. This method has been widely applied to the preparative separation of ionized compounds including various kinds of amino-acid and peptide derivatives, alkaloids, índole auxines,and dyes. Separations of highly polar analytes such as free peptides, catechol-amines, and sulfonic dyes require a ligand such as di-[2-ethylhexyl] phosphoric acid (for basic analytes) and dodecylamine (for acidic analytes), while the resolution of enantiomers necessites introduction of a suitable chiral selector in the stationary phase [4]. Typical examples of pH-zone-refining CCC separations will be presented. Although the method is only applicable to the separation of a relatively large quantity of ionized compounds, it provides the following several important advantages over the conventional CCC: 1) sample loading capacity is increased over 10 times; 2) fractions are highly concentrated near its saturation level; 3) yields of pure fractions are improved by increasing a sample size; 4) charged minute compounds are concentrated and detected at the peak boundaries; 5) elution peaks are monitored with a pH flow meter for the compounds with no chromophore.

References

1. Y. Ito, Y. Shibusawa, H.M. Fales and H.J. Cahnmann, J. Chromatogr. 625 (1992) 177. 2. A. Weisz, A.L. Scher, K. Shinomiya, H.M. Fales and Y. Ito, J. Am. Chem. Sci. 116 (1994) 704. 3. Y. Ito and Y. Ma, J. Chromatogr. A 753 (1996) 1-36. 4. Y. Ma, Y. Ito and A. Foucault, J. Chromatogr. A, 704 (1995) 75-81.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 K-2 Scalable Technology for the Extraction of Pharmaceuticals (STEP): Outcomes from a 3 year Collaborative Research Programme Ian Sutherland1,*, Peter Hewitson1, Lee Janaway2, Philip Wood2, Neil Edwards2, David Rooke2, Guy Harris2, David Keay2, Keith Freebairn3, David Johns3, Nathalie Douillet3, Chris Thickitt3, Clive Mountain3, Ben Mathews4, Roland Brown4 and Svetlana Ignatova1

1,* Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UK, Tel: +44(0)1895 266920, Fax:+44(0)1895 274608, [email protected] 2 Dynamic Extractions Ltd, 890 Plymouth Road, Slough, SL1 4LP, UK 3 GlaxoSmithKline, Pharmaceuticals R&D Facility, Stevenage, SG1 2NY, UK 4 Pfizer, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK

Keywords: Pharmaceuticals, Liquid-liquid Extraction, Scale-up, Counter-current Chromatography, High Value Manufacturing, Solvent Systems

Professor Sutherland will give a general overview of the industrial scale up of countercurrent chromatography and, in particular, will be reporting on the outcome of a 3 year £1.5m Technology Strategy Board (TSB) funded research programme first introduced at CCC2010 [1] developing a small footprint, versatile, counter current chromatography purification technology and methodology which can be operated at a range of scales in both batch and continuous modes and that can be inserted into existing process plant and systems. Our consortium, integrates technology providers (Dynamic Extractions) and the scientific development team (Brunel) with end user needs (GSK & Pfizer), addressing major production challenges aimed at providing flexible, low capital platform technology driving substantial cost efficiency in both drug development and drug manufacturing processes. Advances in process automation, method development, continuous processing, robustness/ reliability and throughput will be presented with scale up examples taken from the 85% success rate with end user applications. While an overview will be given here, delegates will get the opportunity to discover more specific presentations on specific aspects of the TSB-STEP research prorgramme on new solvent systems for rapid method development (Svetlana Ignatova and possibly Guy Harris); continuous processing of waste streams (Svetlana Ignatova) and Intermittent Counter-current Extraction (ICcE – Peter Hewitson) References

[1] Sutherland, I.A., Ignatova, S., Hewitson, P., Janaway, L., Wood, P., Edwards, N., Harris, G., Guzlek, H., Keay, D., Freebairn, K., Johns, D., Douillet, N., Thickitt, C., Vilminot E. and Mathews, B., Scalable Technology for the Extraction of Pharmaceutics (STEP): the transition from academic knowhow to industrial reality, Journal of Chromatography A 2011, 1218, 6114-6121

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 K-3 Terminology for Countercurrent Chromatography

Alain Berthod

Laboratoire des Sciences Analytiques, Université de Lyon, CNRS UMR5180, Bâtiment CPE, 69622 Villeurbanne cedex, France

Corresponding author e-mail: [email protected]

Keywords: Countercurrent chromatography, countercurrent separation, terminology.

Countercurrent chromatography (CCC) is a liquid chromatography technique that uses a support-free liquid stationary phase. A biphasic liquid system is used, one phase of the system is selected to be the mobile phase and the other is the stationary phase. It is difficult to maintain the volume of a liquid stationary phase stable; centrifugal fields are used. The CCC columns must be able to generate this field. They cannot be a simple tube with frits at both ends.

Ever since Yochiro Ito reported the separation of blood plasma cells with a sealed helical tube in 1966, counter current chromatography (CCC) has been a fertile ground for instrumental and technical innovation. Before the birth of hydrodynamic countercurrent separation, the principle of continuous liquid-liquid separation had been demonstrated by liquid-liquid chromatography (LLC) and countercurrent distribution (CCD). The key innovation of CCC was to use a centrifugal force to retain the stationary liquid phase in the column in such a way that it can interact dynamically with mobile phase. The broad diversity of countercurrent separation terminology reflects the innovative spirit of the field as well as the global appeal of this technique.

Rotors, gears, spools, rotating seals are very specific things that are not needed in classical liquid chromatography with a solid stationary phase. The special terminology used in CCC is explained sorting the terms in two classes: the terms linked to the CCC instrumentation and the terms coming form the biphasic liquid system. All the terms common to classical chromatography are not listed.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 K-4 API recovery from Pharmaceutical Waste Streams by High Performance Countercurrent Chromatography and Intermittent Countercurrent Extraction Svetlana Ignatova1,*, Peter Hewitson1, Philip Wood2, Nathalie Douillet3, Chris Thickitt3, David Johns3, Keith Freebairn3, Roland Brown4 and Ian Sutherland1 1,* Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UK, Tel: +44(0)1895 266911, Fax:+44(0)1895 274608, [email protected] 2 Dynamic Extractions Ltd, 890 Plymouth Road, Slough, SL1 4LP, UK 3 GlaxoSmithKline, Pharmaceuticals R&D Facility, Stevenage, SG1 2NY, UK 4 Pfizer, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK

Keywords: Waste Streams, Liquid-liquid Extraction, Scale-up, Counter-current Chromatography, Intermittent Countercurrent extraction, Continuous Processing When new projects are scaled up from the research and development stage into manufacturing, recovery or disposal of waste streams becomes more and more important to minimise cost and reduce both the environmental and safety impact. Modern telescopic synthesis techniques and their follow-on purification processes are not always efficient enough to prevent loss of active pharmaceutical ingredients (API) in the waste stream. There are technologies available by which components of such streams can be recovered, treated or disposed of. However, most of them are based on selective filtration and aim to recover catalysts or reduce toxicity of pharmaceutical waste streams before they are released into the general water supply or burnt. For any waste stream purification, the following stages of process optimization should be considered: 1) prevention and minimising of environmental impact through process design; 2) reuse of materials with no additional processing; 3) recycling materials after such processing if required; 4) disposal with energy recovery and finally 5) benign disposal. This study is addressing 2 & 3. The drive towards greener processes, process economics, technology availability, and legislation, all play a part in the selection of the best available technology for waste stream processing. One such technology is High Performance Counter Current Chromatography (HPCCC) which is being evaluated as new cost effective continuous platform technology as part of a 3 year Technology Strategy Board High Value Manufacturing initiative entitled “Scalable Technology for the Extraction of Pharmaceuticals” [1-2]. This is an industry led collaborative project involving end-users, GSK and Pfizer, a technology supply company, Dynamic Extractions, and Brunel University all actively working together to improve process efficiency and reduce both cost and waste. HPCCC method development for waste steam purification is done at the analytical scale with subsequent scale up to the preparative 1L instrument. This demonstrates the potential of the technology for the recovery of active pharmaceutical ingredients from complex waste streams in both batch and continuous processing modes. The scalability of the HPCCC process enables optimization work at the laboratory scale to be transferred directly to the 18L pilot scale HPCCC. Different scenarios of throughput/purity/yield will be presented demonstrating the flexibility of the technology to purify the target compound at the required trial grade and avoiding expensive clean up processes. This process is therefore capable of reducing the environmental impact of pharmaceutical waste streams. References 1. Technology Strategy Board (TSB) High Value Manufacturing Award (Grant No. TP14/HVM/6/I/BD506K) “Scalable Technology for the Extraction of Pharmaceuticals (STEP) September 2009 to August 2012 2. Sutherland, I.A., Ignatova, S., Hewitson, P., Janaway, L., Wood, P., Edwards, N., Harris, G., Guzlek, H., Keay, D., Freebairn, K., Johns, D., Douillet, N., Thickitt, C., Vilminot E. and Mathews, B., Scalable Technology for the Extraction of Pharmaceutics (STEP): the transition from academic knowhow to industrial reality, Journal of Chromatography A 2011, 1218, 6114-6121

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-1 A new generation of Countercurrent Separation Technology

Samuel Pro1, Tom Burdick1, Luke Pro1, Warren Friedl1, Nick Novak1, Feng Qiu2, Jim B. McAlpine2, J. Brent Friesen2,3, and Guido F. Pauli2,* 1Wrightwood Technologies Inc., Chicago, IL 60660; 2MCP & ITR, University of Illinois, College of Pharmacy, 833 S. Wood St., Chicago, IL 60612, USA; 3Dept. of Physical Sciences, Rosary College of Arts and Sciences, Dominican University, River Forest, IL 60305, USA * [email protected] fax +1 312 355 2693

Keywords: Countercurrent separation, real-time monitoring, phase sensor, K-based separation

Countercurrent separation (CS) methodology has deep roots in natural products and pharmaceutical research and is based on a unique physiochemical mode of separation. We introduce a novel liquid-only LC technology which pioneers integration of all components, volumetrics, ease-of-use, and full automation. The CherryOne instrument is the first CS instrument capable of real-time (RT) monitoring, RT metering, and RT controlling of the dynamic CS liquid-liquid process.

Enabling technology is the newly developed RT phase monitor, which utilizes a high-pressure, non-intrusive permittivity sensor and provides three levels of RT control in CS: (1) RT phase monitoring detects the balance of the column’s stationary and mobile phases (Sf), and also provides input for volumetric and flow control regimes. (2) RT measurement of the partition-coefficient (K). This permits full normalization of the chromatographic x-axes in CS. As a consequence, this enables K-based and targeted purification of analytes with known K values. Utilization of modern CS operation modes, such as EECCC, allow for the determination of K values for the complete range of analytes. (3) RT mixing of biphasic solvent systems with a five-headed delivery system makes possible the direct formulation of HEMWat and related systems. This reduces waste, increases the versatility of any CS column, and improves the already green aspect of CS technology compared to solid-phase LC.

The new object oriented data system (Corbel) of the CherryOne is highly accessible, allows internet-based remote instrument operation, and is compatible with all major CS centrifugal column designs. Applications developed for the targeted purification of botanical reference materials will be presented and demonstrate the power of RT control in CS for natural products and pharmaceutical analysis.

Figure 1. The CherryOne instrument with integrated Corbel data system provides full real-time control over all essential CS parameters such as Sf and K, and enables automated and targeted K-based analysis.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-2 A small volume hydrostatic CCC column for full and quick solvent selection Alain Berthod, Karine Faure, Jeremy Meucci, Nazim Mekaoui Institut des Sciences Analytiques, University of Lyon, Villeurbanne, France *fax +33 472 448 319, e-mail: [email protected]

Keywords: instrumentation, centrifugal partition chromatography, small volume, solvent systems

The two major problems in countercurrent chromatography (CCC) are first to find the appropriate biphasic liquid system for the desired purification and second to retain enough liquid stationary phase inside the CCC column so that the separation is doable. A new 38 mL volume hydrostatic CCC column is presented showing excellent liquid stationary phase retention even for the aqueous two-phase systems most difficult to work with. The small column volume allows for a very fast estimation of the separation properties of a biphasic liquid system.

Table 1 compares the retention volumes and times obtained with two CCC columns of different volumes. The gain in experimental duration is obvious 12 min compared to 200 min (or 3 hours and 20 min). It is due first to the small column volume and second to the higher possible flow rate due to a better hold of the liquid stationary phase. Its usefulness was demonstrated in the direct measurement of octanol/water partition coefficients.

Table 1. Comparing characteristics of CCC columns

Small CCC Regular CCC

Column Vol. 38 mL 250 mL

Vs Sf=60% 23 mL 150 mL

Vr of K = 2 61 mL 400 mL 12 min @ 200 min @ tr of K = 2 5 mL/min 2mL/min

Figure 1. Two hydrostatic CCC columns: left, the Kromaton FCPC 250 that can hold different rotors including the 36 mL rotor; right, the Rousselet-Robatel-Decisive-Design prototype (R2D2), a 38 mL hydrostatic column. The small inset picture gives the prototype scale.

The hydro static rotor was mounted in two different cases: one large case was able to host different rotor sizes (between the smallest 30 mL rotor up to a 1 L rotor). A second instrument was designed around the small rotor minimizing size; the Rousselet-Robatel-Definitive-Design (R2D2) instrument contained the small hydrostatic rotor and demonstrated similar capabilities when compared to the larger and more versatile instrument (Figure 1).

Obviously, the small volume instruments will only be able to purify mg amounts of materials, but the obtained results can be easily transposed to bigger CCC columns of the hydrostatic as well as hydrodynamic type for larger loads using the same biphasic liquid system.

References

K. Faure, N. Mekaoui, J. Meucci, A. Berthod, LC/GC USA, in press, 2012.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-3 Construction of a HSCCC apparatus with column capacity of 12 or 15 liters and its application as flash countercurrent chromatography in quick preparation of (-)-epicatechin 1Qingbao Du, 2Heyuan Jiang, 1，2Junfeng Yin, 1，2Yongquan Xu, 1Wenkai Du, 1Bo Li, 1Qizhen Du* 1Institute of Food Chemistry, Zhejiang Gongshang University, Hangzhou 3100125, China 2 Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China * Corresponding author. Tel./fax: +86 571 88071024 8575. E-mail address: [email protected] (Q. Du).

Keywords: HSCCC; scale-up; FCCC; (-)-epicatechin gallate; (-)-epicatechin; preparation Because most of the so-called HSCCC reported separations were achieved with a relatively long timeframe, Du Recently suggested a term ‘‘Flash counter-current chromatography’’ (FCCC) which is defined as a real separation possessing a characteristic that the flow rate of mobile phase (ml) is equal or greater than the square of the diameter 2 of the column tubing (mm), i.e. Fc/d ≥1, here d and Fc are the diameter of the column tubing and the applied flow rate of mobile phase, respectively. FCCC separation finishes less than 5 h as the tubing length of column is less than 150 m no matter how size of the tubing bore. Thus, FCCC is suitable for scale-up for industrial separation. Presently, an 18-l Maxi-CCC apparatus is scale-ed by Dynamic Extractions. It has two bobbins, symmetrically mounted, of 9-l capacity each which can be operated in parallel or series or as individual bobbins. This apparatus can been used for FCCC separation. However, in laboratory studies, HSCCC instruments with three columns in series occupy more than 70% since successful commerce of Pharma Tech (USA) and Taotou (China). HSCCC instruments with three columns in series possess more compact centrifuge than those instruments with two columns in series. Therefore, scale-up of HSCCC instruments with three columns in series is worth of try. The present study designs two sets of columns with tubing bore of 10 mm and 12 mm yeilding 12 l and 15 l column capacity for a comparative investigation of isolating (-)-epicatechin gallate (ECG) from tea extract by FCCC. Then, hydrolyzing the ECG fraction from FCCC for the separation of (-)-epicatechin (EC) by running flash column chromatography on macroporous resin AB-8 resulted in quick preparation of EC. The column of 12 l composed of three 4 l-columns in series. The tubing I.D. is 10 mm and βvalue ranges 0.55-0.75. The apparatus was used for the separation of epicatechin gallate (ECG) from tea extract by flash countercurrent chromatography using a two-phase solvent system composed of hexane-ethyl acetate-water (1:2.5:4). In each separation, a sample amount of 200 g of the tea extract could be loaded to give 7.5 l of water solution containing 39 g of ECG without peaks of impurities. The column of 15 l composed of three 5 l-columns in series. The tubing I.D. is 12 mm and βvalue ranges 0.50-0.75. Under the same condition, the apparatus could load a sample amount of 200 g of the tea extract to yield 9.0 l of water solution containing 40 g of ECG without peaks of impurities.

Fig. 1. Chromatograms of the tea extract on preparative FCCC system. Samples: 200 g in 1600 ml mobile phase; rotation speed: 600 rpm; flow rate: 100 ml/min. Retention of stationary phase: 58%.

Fig. 2. Chromatograms of the tea extract on preparative FCCC system. Samples: 200 g in 1600 ml mobile phase; rotation speed: 600 rpm; flow rate: 150 ml/min. Retention of stationary phase: 56%. 16.5 liters of the ECG solution was hydrolyzed by tannase in 2 hour. The hvdrolysate was purified by flash column chromatography on macroporous resin AB-8 (1kg) to yield 52 g of EC with a high purity of 99.1 %. [1] I.A. Sutherland, J. Chromatogr. A 1151 (2007) 6. [2] Qiao, Q.; Du, Q. Preparation of the monomers of gingerols and 6-shogaol by flash high speed counter-current chromatography. J. Chromatogr. A, 1218 (2011) 6187- 6190. [3] Du, Q. (2011) The features of rapid counter-current chromatographic separations, J. Liquid Chromatogr. Rel. Technol. 34, 2074-2084.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-4 Seady-state and Non-steady state Operation of Counter-current Chromatography devices Artak E. Kostanyan Kurnakov Institute of General & Inorganic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 31, Moscow 119991, Russia fax +495 9554834, e-mail: [email protected]

Keywords: Counter-current chromatography; liquid–liquid extraction

Counter-current chromatography (CCC) is a non-steady state liquid–liquid extraction process. CCC apparatuses can be operated under different conditions: in steady-state (as ultrahigh-efficient extraction columns for counter-current separation processes) and non-steady state (as chromatographic devices for preparative and analytical purposes) regimes. The aim of this report is to analyze and compare different possible variants of separation and purification processes by using steady-state and non-steady state operating modes of CCC columns. Three approaches – the plate theory, the cell model and the Craig model, commonly used for mathematical description of separation processes in CCC and extraction columns, are discussed. The separation of the dual counter-current process conducted in steady-state (fractional extraction) and non-steady state (with impulse and non-impulse sample loading) operating modes are compared with the conventional CCC separation. In the dual counter-current separation, both phases are mobile and flowing in opposite directions in the CCC column; the sample is introduced at the middle of the column and the components to be separated are eluted from opposite ends of the column. In steady-state (counter-current fractional extraction) operating mode, where the sample is fed continuously at a constant rate, the concentration of solutes eluted with both phases become constant after a while. In non-steady state (chromatographic separation) operating mode the sample is fed into the column only during a certain time < run time, and the concentration of solutes eluted with phases as in case of conventional CCC separation are not constant. Basing on the cell model theory equations are presented allowing the simulation of the separation processes for both operating modes. To compare the steady-state extraction and chromatographic separation with impulse sample injection, a special case of extraction, when the mixture to be separated is fed without a solvent, is considered. For this case of extraction, the normalized concentration of solutes eluted with x and y phases are determined by the following formulas: x 1 y F / F X = = Y = = 1 2 x 1+ (1−ϕ )ϕ /ϕ x 1+ϕ /[(1−ϕ )ϕ ] 0 1 2 1 , 0 1 1 2

An1 − An1+1 Bn2 − Bn2+1 ϕ = , ϕ = , 1 1− An1+1 2 1− Bn2+1

F1 F2KD Q A = , B = , x0 = , F2KD F1 F1 where F1 and F2 are the volumetric flow rates of x and y phases, respectively; KD = y/x is the partition coefficient; n1 and n2 are the numbers of equilibrium cells in the right and left of the column middle; Q is the rate of the solute continuously fed into the middle of the column. It is established that the separation efficiency of steady-state (extraction) and non-steady state (chromatography) dual counter-current processes are absolutely identical and significantly higher than that of the conventional CCC separation. The results obtained demonstrated the potential of the steady-state dual counter-current processes for preparative and industrial-scale separations. Using CCC columns to conduct counter-current extraction processes can allow increasing the productivity by an order of magnitude ensuring, a desirable separation. In interaction with the suppliers, these devices are to be improved to provide a sound, stable operation in the continuous steady-state conditions.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-5 True moving bed CPC for continuous purification of natural substances G.Audo*, C.Le Quémeneur

Armen Instrument – application laboratory – ZI Kermelin – 16, rue Ampère - 56890 Saint Ave, France Tel : +33 (2) 97 61 84 00; Fax : +33 (2) 97 61 85 00; e-mail : [email protected]

Keywords: TMB, continuous,

Here is presented a comparison between batch CPC and two steps continuous CPC TMB as True Moving Bed method to purify natural substances. Classically, batch injection are done by injecting a define quantity of crude mixture. Size of the CPC column, solubility, mass injected and time of run define the productivity. CPC TMB system allows to inject continuously the sample between two CPC columns and to recover from one side of the system the more polar compounds which have more affinity for lower aqueous phase and on the other side the less polar compounds of the extract which have more affinities for the upper organic phase (Figure 1).

Figure 1. True Moving Bed CPC general flowchart

To obtain pure compounds with CPC TMB two continuous steps are needed to eliminate polar impurities first and then non polar impurities. As continuous process it allows very high productivity with a minimum of operation for low cost processing

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-6 Design of a Continuous Centrifugal Partition Chromatographic Process Johannes Goll, Andreas Frey, Mirjana Minceva* Chair of Separation Science and Technology, Friedrich Alexander University Erlangen-Nuremberg, Germany * Egerlandstraße 3, 91058 Erlangen, Fax: 09131-85-27441, E-Mail: [email protected]

Keywords: sequential centrifugal partition chromatography, mathematical modeling, process design

Sequential centrifugal partition chromatography (sCPC) is a novel continuous cyclic separation process (1). Using sCPC a mixture of components which distribute differently between the two phases of a biphasic liquid system can be separated into two products. One of the specifics of the process is that the feed stream is introduced continuously in the middle of the sCPC unit whereas the two products are collected sequentially in each step of the cycle at the opposite ends of the unit. The two steps of one sCPC cycle differ by the liquid phase used as mobile phase (upper or lower phase) and its flow direction. The process includes several interconnected operating parameters (feed concentration, feed and mobile phase flow rate in each step and the duration of each step of the cycle). Hence, the selection of sCPC unit operating parameters is not easy and straightforward.

In this work a method for the design of a sCPC separation of a binary feed mixture is presented. The objective is to define sCPC unit operating parameters which lead to complete feed separation, maximum productivity and minimum solvent (mobile phase) consumption. The method includes several steps which are strongly supported by mathematical modeling and simulation.

At first, a biphasic solvent system from the ARIZONA solvent system family is selected by screening of the partition coefficients of the feed components using the a priori predictive thermodynamic method “Conductor-like Screening Model for Real Solvents” (COSMO-RS) (2). In the next step, the distribution equilibrium of the feed components in the selected biphasic solvent system is determined by shake flask experiments. The maximum mobile phase flow rate in each sCPC step is derived from a study of the stationary phase retention as a function of the mobile phase flow rate. In the following, the column efficiency is determined by pulse injection experiments. The experimentally determined thermodynamic equilibrium, hydrodynamics and mass transfer parameters are incorporated in a mathematical model, which is used for the simulation of the sCPC operation. The sCPC unit operating parameters, including

feed and mobile phase flow rate and duration of the step time, are selected using the constraints derived in one of our previous publications (3,4). At the end, the maximum feed concentration is determined by simulation of the sCPC operation with a gradual increase of the feed concentration.

The method is demonstrated for a complete separation of a binary mixture of pyrocatechol and hydroquinone. The proposed method is validated by comparison of the experimental and calculated sCPC separation performances. In comparison with the conventional batch operation, the sCPC offers better overall separation performances.

References 1. F. Couillard, A. Foucault, D. Durand, US 0243665 A1, 2006.

2. E. Hopmann, W. Arlt, M. Minceva, J. Chromatogr. A, 1218 (2011) 242. 3. J. Völkl, W. Arlt, M. Minceva, AIChE J, 2012, in press DOI 10.1002/aic.13812. 4. E. Hopmann, J. Goll, M. Minceva, Chem. Eng. & Technol., 35 (2012) 72.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-7 Intermittent Counter-current Extraction: Scale up and an Improved Bobbin Design Peter Hewitson*1, Ian Sutherland1, Philip Wood2, Svetlana Ignatova1 *1 Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UK Tel: +44(0)1895 266917, Fax: +44(0)1895 274608, [email protected] 2 Dynamic Extractions, Ltd, Slough, UK Keywords: Intermittent counter-current extraction, liquid-liquid extraction

Intermittent counter-current extraction (ICcE) has been demonstrated as a potential liquid-liquid extraction method for continuous separation of compounds into two product streams and the isolation, concentration and enrichment of a low abundance target compound, while washing away all other compounds (1). Further the scale-up of the technology using commercial twin-column CCC instruments has been successfully demonstrated (2) and the effect of key operating parameters on both selectivity and throughput has been shown (3). We describe results for the scaling of ICcE from the semi preparative DE-Spectrum instrument (143 mL) to preparative DE- Midi instrument (912 mL) and on to pilot scale Maxi instrument (4.6 L) using a model mixture of compounds from the GUESSmix across a range of polarity hexane/ethyl acetate/methanol/water (HEMWat) phase systems from a polar system (1:4:1:4) to a non polar system (4:1:4:1). These results are compared to a model ICcE to confirm the prediction of compound elution order. With ICcE, the sample is continuously injected between the two columns while the mobile phase flow is intermittently switched between normal and reversed phase modes. When using conventional bobbins, with each switch an inherent delay occurs as a volume of the previous mobile phase is displaced from the flying leads into the fractions. Therefore, a new bobbin design with modified end fittings which allow direct switching of mobile phase flow at the column inlet is described (Figure 1). This design eliminates both the delay and the displaced dead volume which usually occurs with each switch of mobile phase flow and therefore reduces any disruption to the column equilibrium as the previous mobile phase is pumped from the flying leads. These bobbins have been designed so they can operate as standard isocratic columns, if required. Using the same GUESSmix and phase systems results shown a 6% improvement in the retention times, equivalent to the eliminated delay. Further research is ongoing to assess the improvements to the separation efficiency of these columns.

UP Sample

UP Sample Pump Pump UP Detector Column 1 Column 2

Sample LP Detector Pump LP Pump NORMAL PHASE REVERSED PHASE LP Sample

Figure 1: ICcE schematicshowing twin columns with separate inlet and outlet leads for flow of upper and lower phases

References (1) Hewitson, P.; Ignatova, S.; Ye, H.; Chen, L.; Sutherland, I. Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal medicine. Journal of Chromatography A 2009, 1216, 4187-4192. (2) Sutherland, I.; Hewitson, P.; Ignatova, S. Scale-up of counter-current chromatography: Demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale. Journal of Chromatography A 2009, 1216, 8787-8792. (3) Hewitson, P.; Ignatova, S.; Sutherland, I. Intermittent counter-current extraction - Effect of the key operating parameters on selectivity and throughput. Journal of Chromatography A 2011, 1218, 6072-6078.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-8 Selection of the Mobile and Stationary Phase in Support Free liquid-liquid Chromatography applying a Predicitve Thermodynamic Model Andreas Frey, Mirjana Minceva

Chair of Separation Science and Technology, Friedrich Alexander University Erlangen-Nuremberg, Germany

Egerlandstraße 3, 91058 Erlangen Fax: 09131-85-27441 E-Mail: [email protected]* [email protected]

Keywords: Solvent system selection, Partition coefficient, COSMO-RS

The selection of the mobile and stationary phase in support free liquid-liquid chromatography (LLC) is equivalent to a selection of a biphasic solvent system. The limitless choice of solvents requires a systematic method for the selection of the appropriate solvent combination for a given separation task. At the moment, this selection is performed by “trial and error” experimental screening procedures, using only a restricted number of solvents and solvent combinations. In this work a general approach for the selection of the mobile and the stationary phase for a specific LLC separation task is presented. The approach is strongly supported by the use of the a priori predictive thermodynamic method “Conductor-like Screening Model for Real Solvents” (COSMO-RS). (1) In the first step, potential solvents are selected based on criteria defined by the user. The solubility of the target solutes in the selected solvents is evaluated using the solvent capacity, calculated with COSMO-RS. After that, biphasic liquid systems (obtained by mixing of at least three solvents) are chosen from available liquid-liquid phase compositions and diagrams. In a next step, a screening of the pre-selected biphasic liquid systems is performed using the partition coefficient calculated with COSMO-RS. (2) Systems in which the target solute obtains a partition coefficient in a range from 0.4 and 2.5 are selected and further evaluated performing a LLC separation in a small scale unit. At the end the system leading to the best separation performance i.e. the best productivity and the lowest solvent consumption is selected for the given separation task. It is demonstrated that with the proposed approach the extensive experimental effort needed for the selection of the biphasic liquid system can be significantly reduced.

References

1) E. Hopmann, A. Frey, M. Minceva, J. Chromatogr. A 1238 (2012) 68 2)E. Hopmann, W. Arlt, M. Minceva, J. Chromatogr. A 1218 (2011) 242

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-9 Selection and use of aqueous two phase systems in centrifugal partition chromatography Christoph Schwienheer*, Stephan Adelmann, Juliane Merz, Gerhard Schembecker

TU Dortmund, Laboratory of Plant and Process Design * Emil-Figge-Straße 70, 44227 Dortmund, Germany; fax +492317552341; [email protected] CPC, ATPS, flow pattern, operating parameter The separation efficiency in Centrifugal Partition Chromatography (CPC) is strongly influenced by the hydrodynamics in the chambers. Especially flow pattern and the resulting stationary phase hold-up and interfacial area for mass transfer are important parameters. While there are already some experimental results for aqueous organic systems and some hints on appropriate selection of phase system conditions and operation parameters [1,2], the use of aqueous two phase systems (ATPS) in CPC is almost not considered yet. ATPS are obtained from two aqueous solutions containing different polymers ore one polymer and one salt in adequate concentrations. ATPS are gentle for the separation of biotechnological products like proteins or antibodies. Resulting on their physical properties (low interfacial tension, low density difference and high viscosities compared to usual aqueous organic solvent systems) the phase separation can only be achieved poorly. Thus, the use of ATPS in CPC is difficult because there is a strong bleeding of stationary phase [3], resulting in a low hold-up and a low separation performance. To indicate the ability of ATPS in CPC, the physical properties (viscosity and density of both phases and interfacial tension) were measured by varying the molecular weight of polyethylenglycol (PEG) and the concentrations of PEG and phosphate salt. The viscosity increases more with increasing molecular weight than with increasing the concentration of PEG. The density difference and interfacial tension, however, are mainly increased by increasing concentrations of the phase forming components. Based on these results several ATPS were selected for visualization of their hydrodynamical behavior in CPC chambers. Using an optical experimental setup [2] and a phase selective dye for coloring the mobile phase we could observe the flow of the mobile phase and measure the hold up of stationary phase for different operating parameter. As a result of these investigations, the hydrodynamical ability of different ATPS could be determined and appropriate operation parameter could be found. In general, the lower viscous phase of the system should be the mobile phase and the rotational speed of the CPC should be as high as possible. As exemplarily shown in figure 1 it is useful to increase the concentrations of the phase forming components to decrease the bleeding of stationary phase and achieve a higher hold up. Finally, separation experiments were performed for a mixture of exemplary proteins to evaluate the influence of mentioned parameters on the separation efficiency.

Figure 1. Exemplary results for the hold-up of stationary phase measured for different APTS with variation in PEG molecular weight and concentration of phase forming components.

References

1. L. Marchal et.al; AIChE Journal; vol. 48, no. 8; 2002; 1692-1704 2. S. Adelmann et.al; Journal of Chromatography A; vol. 1218, no. 32; 2011; 5401-5413 3. I. Sutherland et.al; Journal of Chromatography A; vol. 1218, no. 32; 2011; 5527-5530

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-10 Modelling Counter-current Chromatography using a Temperature Dependence Plate Model Yun Wei *, Fengkang Wang, Shui Wang *State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China. Tel & Fax: 0086 10 64442928. E-mail: [email protected] Keywords: Counter-current Chromatography, partition coefficient K values, temperature, modelling

ABSTRACT Recently research workers have built several math models of counter-current chromatography (CCC), such as cell model1, CCC distribution model2, Continuous-Stirred Tank Reactors model3, probability model4. A new math model of counter-current chromatography using temperature dependence plate model has been described in this paper. Based on plate theory and van’t Hoff equation, a new temperature dependence plate model has been set up. The math modelling of the partition action of CCC can be achieved by using Matlab to do computer programming. The relationship between temperature and partition coefficient K value can be summarized into linear equations (logK=A-B/T), which can be introduced to the plate model. K values decrease as temperature getting higher. R values are related to K values, as a result, temperature change finally leads to resolutions between different compounds change. K values at different temperatures of a certain compound in a given system were calculated according to those equations above. New program with temperature parameter was applied in prediction of experimental result, which decreased the error in modelling prediction. The retention time error is 9.22% for peak 2 and 5.46% for peak 4 (fig.1), respectively, without considering temperature effect. When temperature was taken into consideration, the error was significantly reduced (fig.2). This temperature dependence plate model can well explain why CCC partition efficiency is not the same in different seasons without temperature controller, and it can be more accurate in CCC separation prediction than previous models. As we all know, two compounds can’t be separated when the separation factor is 1. However, if their logK-1/T equations are different, separation can be achieved by changing temperature and the optimal temperature can be obtained from this new model. Those logK-1/T equations may be applied to other models in the future.

0.025 0.025 experimental result simulated 2 experimental result 0.020 simulated 2 simulated 4 0.020 simulated 4

0.015

0.015

0.010 0.010 2 2 0.005 4 mg/ / malsed bance e c n a rb o s b A d e lis a rm o )/N L /m g (m c n o C 0.005 4

n( / )Nomaie bsorbance A alised orm L)/N g/m onc(m C 0.000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 0.000 t/h 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 t/h

Fig.1. Black line: chromatogram of the crude extract from Flaveria bidentis (L.) Kuntze by HSCCC. Solvent system: ethyl acetate–methanol–water 50:1:50 (v/v); stationary phase: upper Fig.2. Black line: experimental result organic phase, mobile phase: lower aqueous phase, flow rate: 2.0 ml/min, revolution speed: 800 rpm, sample: 400mg Red line: simulated peak 2 at 291.15K \ dissolved in 10 ml lower phase. Peaks: 2 was patuletin-3-O-glucoside, 4 was astragalin. Red line: Blue line: simulated peak 4 at 296.35K simulated peak 2 not considering temperature effect Blue line: simulated peak 4 not considering temperature effec

References 1. Kostanian, A. E. J. Chromatogr. A 2002, 973, 39-46. 2. Sutherland, I. A.; Folter, J. de. ; Wood, P. J. Liq. Chromatogr. Related Technol. 2003, 26, 1449-1474. 3. Guzlek, H.; Baptista, I.I.R.; Wood P. L.; Livingston, A. J. Chromatogr. A, 2010, 1217, 6230–6240. 4. Folter, J. d.; Sutherland I. A. J. Chromatogr. A, 2011, 1218, 6009-6014.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-11 Modeling Gradient Elution in CCC: Efficient Separation of Tanshinones S. Wu1 D. Wu1 J.Liang1 A. Berthod2 1-Siyuan research center in Pharmacy and Biotoxicology, College of Life Science, Zhejiang University, Hangzhou 310058, China 2-Institut of Analytical Sciences, CNRS, University of Lyon, 69622 Villeurbanne, France *fax +33 472 448 319; e-mail: [email protected]

Keywords: Liquid systems, gradient elution, theory, tanshinones

Counter-current chromatography (CCC) is a support-free liquid-liquid chromatography using centrifugal fields to hold the liquid stationary phase. CCC has been widely applied in the separation of various natural and synthetic components using a variety of biphasic liquid systems. The related hexane or heptane/ethyl acetate/methanol or ethanol/water biphasic liquid systems (HEMWat or HEEWat systems, respectively) demonstrated their significance in CCC. Gradient is difficult in CCC since any composition change in one phase induces a composition change of the other phase to maintain phase equilibrium. This work provides a new insight into linear gradient elution in CCC that is feasible with some biphasic liquid systems such as selected compositions of the HEMWat or HEEWat systems. Two approaches are presented and compared: the first approach proposes the equations modeling solute motion inside the CCC column; the second approaches consider the gradient mobile phase composition change using a step by step method. The two views are compared in terms of predicted solute positions. Particular compositions of the HEEWat liquid system, namely the hexane/ethyl acetate/ethanol/ water 8:2:E:Wat compositions with E+Wat = 10, were experimentally studied from Wat = 1 to Wat = 9. They showed moderate changes in the upper organic phase compositions. The two model are tested with the separation of tanshinones from the rhizome of Salvia miltiorrhiza Bunge. Different linear solvent gradient profiles were experimentally performed between 8:2:5:5 and 8:2:3:7 HEEWat compositions and the results were evaluated using the two proposed models. Five tanshinones including dihydrotanshinone I, cryptotanshinone, tanshinone I, 1,2-dihydro tanshinquinone and tanshinone IIA have been successfully separated (>95% purities). The gradient models can be used only with biphasic liquid systems in which one phase shows minimum composition changes when the other phase composition changes notably. This case is not the general case for biphasic liquid systems but can be applied with specific compositions of the quaternary HEMWat or HEEWat most useful CCC liquid systems. UV absorbance 220280 nmnm (a.u.)

A Figure 1: Gradient CCC separations a crude

5 extract of S. miltiorrhiza Bunge. The separation 1 4 is done with the HEEWat solvent system 8:2:5:5 2 3 for A: 250 min and B: 200 min and next, the mobile phase is linearly changed to the lower

0 70 140 210 280 350 420 480 aqueous phase of the 8:2:7:3 HEEWat system in A: 150 min and B: 100 min. Compounds 1, B dihydrotanshinone I; 2, cryptotanshinone; 3, tanshinone I; 4, 1,2-dihydrotanshinquinone; 5, 5 4 tanshinone IIA. CCC conditions: flow rate: 2 1 2 3 mL/min; rotation speed: 850 rpm; temperature: 30oC; sample loading: 200 mg injected in 10 mL; elution mode: head-to-tail elution direction. Initial 0 70 140 210 280 350 420 Time (min)

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-12 Investigation of hydrodynamics in CPC chambers using image processing and computational fluid dynamics Juliane Merz*, Christoph Schwienheer, Stephan Adelmann, Gerhard Schembecker

TU Dortmund, Laboratory of Plant and Process Design * Emil-Figge-Straße 70, 44227 Dortmund, Germany; fax +492317552341; [email protected] Keywords: CPC, flow pattern, operating parameter

Centrifugal partition chromatography (CPC) is a kind of liquid-liquid partition chromatography in which the separation mechanism is based on the principle of different distribution of components between two immiscible liquids. Using a centrifugal force, one phase is kept stationary (stationary phase) in chambers while the other phase (mobile phase) is pumped through the stationary one. The efficiency of separation by using CPC devices is strongly influenced by the hydrodynamics in the chambers. Especially flow pattern and the resulting stationary phase hold-up and interfacial area for mass transfer are important parameters. Their relation on physical properties of solvent systems, operating parameters and chamber geometry are not completely understood yet. Only a few research groups have developed a possibility to observe the flow of mobile phase through the stationary phase in the rotating chambers [1]. In order to receive a more detailed impression of flow pattern and derived parameters like stationary phase hold-up and interfacial surface area, we have developed a new method for flow visualization. The developed optical measurement device is attached to a conventional FCPC® unit by Kromaton. We use a single disc covered by acrylic glass, in order to look inside the chambers. To make the flow of mobile phase visible, it is colored with a phase selective dye. The dye absorbs the light of a LED installed at the bottom of the apparatus and pictures were taken by a monochromatic CCD camera installed at the top (see figure 1). Both, LED and camera are triggered by a fork light barrier, to achieve one picture per revolution. After some image processing using customized programs, high contrast images of the flow pattern and values for hold up of stationary phase and interfacial surface area are achieved. [2] Using this optical measurement device, the flow pattern and hold-up for a large variation of common two phase systems in dependency of operation conditions (volume flow of mobile phase, revolution speed, mode of operation) was investigated. Further research is carried out with aqueous two phase systems, for which coalescence of mobile phase and the resulting hold up of stationary phase have to be optimized. Additionally, new CPC chambers for more efficient separations are investigated by using the optical measurement device and additionally computational fluid dynamics as presented by Adelmann et.al. [3].

Figure 1. Sketch of the optical measurement device setup.

References

1. L. Marchal et.al; AIChE Journal; vol. 48, no. 8; 2002; 1692-1704 2. S. Adelmann et.al; Journal of Chromatography A; vol. 1218, no. 32; 2011; 5401-5413 3. S. Adelmann et.al; Journal of Chromatography A; vol. 1218, no. 36; 2011; 6092-6101

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-13 Research of Interaction between Ionic Liquid and Tea Polyphinols by computer simulation assisted High-performance Counter-current Chromatography Chen Xiao-Fen1,2, Huang Xin-Yi1, Wang Gao-hong1,2, Zhang Jia1, Di Duo-Long1*

1 Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000 2 Graduate University of the Chinese Academy of Sciences, Beijing 100049 *Corresponding author fax: 0931-4968094; e-mail: [email protected]

Keywords: Ionic liquid; high-performance counter-current chromatography; tea polyphenols

Ionic liquids (ILs) are entirely composed of cations and anions, which have unique properties such as environmental benefits, nonvolatility, noninflammability, high thermal stability and designability. In recent years, it seemed to ILs in high-speed counter-current chromatography (HSCCC) has attracted scientists’ attention. However, to date, there are no reports that discussed the mechanism about ILs applied to HSCCC.

The polyphenols, (-)-epigallocatechin-3-gallate (EGCG), (-)-gallocatechin gallate (GCG) and (-)-epicatechin-3-gallate (ECG) (Figure 1), were main biological ingredients in tea leaf and possessed highly similar structure. In this study, they were chosen as model moleculars to study the effect of ionic liquid as solvent additives in HPCCC separation.

Basic solvent system of ethyl acetate- water (5:5, v/v) was employed. It was found that three target compounds were inclined to distribute in upper phase. As the increase of hydrophilic ionic liquids added in solvent system, they were draw to lower phase due to the attraction of ionic liquids, which indicated by the decrease of K values (Figure 2). It was planned to combine computer simulation and spectroscopy methods with HPCCC to illustrate the interaction between ionic liquids and target compounds.

Figure 1. Structures of Figure 2. The effect of the concentration polyphenols, A: EGCG, B: of 1-methyl-3-methylimidazolium ECG, C: GCG. tetrafluorobarate on K-values in ethyl acetate–water (5:5, v/v).

Acknowledgements: Authors gratefully acknowledge the financial support by the ‘Hundred Talents Program’ of the Chinese Academy of Sciences (CAS), the National Natural Sciences Foundation of China (NSFC No. 20974116, 21175142).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-14 Modeling of step wise gradients in HSCCC; A rapid and economical strategy for one step separation of eight dihydropyranocoumarins from Seseli resinousm.

Omer Shehzad1, Salman Khan1, In Jin Ha1, Kyoung Jin Lee1, Alev Tosun2, Yeong Shik Kim1*

1Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Korea 2 Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Tandoğan, Ankara, Turkey * Fax: +82 2 765 4768599. E-mail: [email protected] (Prof. Yeong Shik Kim). Keywords: Step wise gradients; Seseli resinosum; 8 Coumarins; HSCCC-ELSD.

Target purification of compounds with a broad polarity range from traditional medicinal plants is a big challenge for counter-current chromatography (CCC). Once a suitable solvent system is selected, the separation process requires optimization of various adjustable parameters. With the development of separation science, gradients elution was introduced in CCC. Common gradient systems in CCC include temperature gradient, flow gradient, linear solvent gradients, pH-gradient and a salting-out gradient of the mobile phase components. In the present study, various operational parameters were optimized to pair the flow rate and solvent gradients for quick isolation of eight dihydropyranocoumarins from the hexane fraction of Seseli resinosum in a single run. For ensuring the high purity of each compound only the main peaks were collected while all the shoulder peaks were mixed and isolated separately by using the same conditions with semi preparative HSCCC. Meanwhile, for the economical aspect and environmental safety the two phase solvent system was prepared using an on-demand solvent preparation mode where the components of each phase were analyzed by GC-FID. The separation efficacy was further enhanced by increasing the system temperature from 15 0C to 35 0C, that lead to decrease in operation time and increase in stationary phase retention. This separation process produced eight dihydropyranocoumarins; [1] d-laserpitin, [2] (3´S,4´S)-3´-angeloyloxy-4´-hydroxy-3´,4´-dihydroseselin, [3] samidin, [4] (3´S,4´S)-3´-acetoxy-4´-angeloyloxy-3´,4´-dihydroseselin, [5] corymbocoumarin, [6] calipteryxin, [7] (3´S,4´S)-3´, 4´-disenecioyloxy-3´,4´-dihydroseselin, (3´R,4´R)-3´, and [8] anomalin. Molecular structures of these compounds have been identified by electrospray ionization mass spectrometry (ESI-MS), one- and two-dimensional nuclear magnetic resonance (1D- and 2D-NMR). This method will allow us to generate a strategy for target separation of compounds with a broad range polarity from any natural product.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-15 Isolation of bioactive polyphenols from by-products of food processing using countercurrent chromatography

P. Winterhalter*, G. Jerz, S. Kuhnert, A. Juadjur, S. Macke

*Technische Universität Braunschweig, Institute of Food Chemistry, Schleinitzstrasse 20,

38106 Braunschweig, Germany (Fax: +49-531-3917200, email: [email protected])

*Correspondence address marked with asterisk

Keywords: Bioactives, Anthocyanins, Copigments, Procyanidines, Stilbenes, Preparative CCC

Strategies for the large-scale isolation and fractionation of polyphenolic extracts from waste material will be demonstrated. Examples are the preparation of bioactive by-products from side streams of the wine and fruit juice industry (1,2). The combination of countercurrent chromato-graphy with membrane techniques allows the separation of crude extracts in the following three groups of polyphenols: anthocyanins, colorless copigments (e.g. flavonols, flavanols, cinnamates, stilbenes) and polymers. By using a novel membrane technology, a pure anthocyanin fraction free of other copigments and polymeric phenols can be obtained (3). For further isolation of bioactives from the copigment fraction, countercurrent chromatography is applied. Techniques that have been applied include inter alia low speed rotary countercurrent chromatography (LSRCCC) and spiral-coil low speed rotary countercurrent chromatography (Spiral-coil LSRCCC). With regards to the polymeric constituents, especially the procyanidines, a depolymerization process will be presented converting the high molecular compounds in bioavailable ones (dimers and trimers) (4-6). Finally initial results concerning the use of grape vine shoots for the preparation of highly bioactive oligomeric stilbenes and their subsequent separation using countercurrent chromatography will be presented (7). These examples demonstrate the need for preparative CCC-systems in order to supply the bioactives in sufficient amounts for the food and pharmaceutical industry.

References

1. M. Schwarz, S. Hillebrand, S. Habben, A. Degenhardt, P. Winterhalter, Application of high-speed countercurrent chromatography to the large-scale isolation of anthocyanins. Biochem. Engineering J. 14, 179-189 (2003).

2. D. Cooke , M. Schwarz, D. Boocock, P. Winterhalter, W.P. Steward, A.J. Gescher, T.H. Marczylo, Effect of cyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model of intestinal carcinogenesis - Relationship with tissue anthocyanin levels. Int. J. Cancer 119, 2213-2220 (2006).

3. A. Juadjur, P. Winterhalter, Development of a novel adsorptive membrane chromatographic method for the fractionation of polyphenols from bilberry. J. Agric. Food Chem. 60, 2427-2433 (2012).

4. N. Köhler, V. Wray, P. Winterhalter, Preparative isolation of procyanidins from grape seed extracts by high-speed counter-current chromatography. J. Chromatogr. A 1177, 114-125 (2008).

5. T. Esatbeyoglu, P. Winterhalter, Preparation of dimeric procyanidins B1, B2, B5, and B7 from a polymeric procyanidin fraction of black chokeberry (Aronia melanocarpa). J. Agric. Food Chem. 58, 5147-5153 (2010).

6. T. Esatbeyoglu, V. Wray, P. Winterhalter, Dimeric Procyanidins: Screening for B1 to B8 and semisynthetic preparation of B3, B4, B6, and B8 from a polymeric procyanidin fraction of white willow bark (Salix alba). J. Agric. Food Chem. 58, 7820-7830 (2010).

7. S. Macke, G. Jerz, M. Empl, P. Steinberg, P. Winterhalter, Activity-guided isolation of resveratrol oligomers from grape vine using countercurrent chromatography, submitted to J. Agric. Food Chem.

Financial support by the Federal Ministry for Education and Research (grant 0315373) is gratefully acknowledged.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-16 Study on preparative separation and purification of chemical compounds from plants by hsccc

Chen Xiao-Fen1,2, Huang Xin-Yi1, Wang Xiao-fei1, Shi Ming-rui1, Wang Gao-hong1,2, Zhang Jia1,3, Di Duo-Long1,3*

1 Key laboratory of Chemistry of Northwestern Plant Resources, Chinese Academy of Sciences & Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy ofSciences, Lanzhou 730000 2 Graduate University of Chinese Academy of Sciences, Beijing 100049 3 Department of Pharmacy，Gansu College of Traditional Chinese Medicine，Lanzhou 730000 *Corresponding author fax: 0931-4968094; e-mail: [email protected]

Keywords: High speed countercurrent chromatography; high shear technique; ionic liquid; purification; preparation;

In recent years, High-Speed Countercurrent Chromatography technique (HSCCC) has been broadly applied for the preparative separation and purification of chemical components from plants due to its unique advantages. In this paper, advancements on application of HSCCC in separation and purification of active compounds from plants are present by our research group as follows:

1. Preparative separation and purification of highlypolarity and thermolabile components from the rhizome of Sphallerocarpus Gracilis and the leaves of Stevia rebaudiana Bertoni by HSCCC, respectively.

2. Screening and preparative separation of active components from the leaves of Oleaeuropaea L. and the rhizome of Radix Astragali by HSCCC, respectively.

3. New Way development: Online extraction and .preparative separation of chemical constituents from Brassica napus L. pollen by high shear technique (HSDE) coupled with HSCCC

4. Study on ionic liquids (RTIL) as the modifier in two-phase solvent system of HSCCC on preparative separation of chemical components from Brassica napus L. pollen and Tea.

5. Study on the surface modification and separation mechanism of PTFE column of HSCCC.

Acknowledgements: The authors gratefully acknowledge financial support by the ‘Hundred Talents Program’ of the Chinese Academy of Sciences (CAS), the National Natural Sciences Foundation of China (NSFC No. 20974116, 21175142).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-17 Enantioseparation of 2-phenylpropionic acid by recycling high speed counter- current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector Ye Zheng，Shengqiang Tong*，Jizhong Yan College of Pharmaceutical Science, Zhengjiang University of Technology, Hangzhou 310032, China *Corresponding author.Tel.:+86 571 88320613; fax: +86 571 88320913 E-mail addresses: [email protected] Keywords: Chiral separation; High speed counter-current; chromatography; Hydroxypropyl-β-cyclodextrin; 2-Phenylpropionic acid

Abstract: Recycling high speed counter-current chromatography (HSCCC) was successfully applied to resolution of 2-phenylpropionic acid enantiomer using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol-1 phosphate buffer solution with pH=2.67 (8:2:10, v/v/v) was selected. Influence factors involved in the chiral separation were investigated, including the concentration of HP-β-CD, pH value of the aqueous phase, the separation temperature. Suitable elution mode was selected for HSCCC enantioseparation of (±)-2-phenylpropionic acid. Under optimum separation conditions, 50 mg of (±)- 2-phenylpropionic acid was separated using preparative recycling HSCCC with multiple recycling elution mode. The purities of both of the fractions including (+)-2-phenylpropionic acid and (-)-2-phenylpropionic acid from the preparative CCC separation were over 92.5% determined by HPLC.

Fig.1. Separations of (±)-2-phenylpropionic acid by preparative chiral HSCCC with recycling elution mode. Experimental conditions: solvent system: n-hexane-ethyl acetate-0.1 mol/L phosphate salt buffer solution with pH=2.67 (8:2:10, v/v/v) containing 0.10 mol/L HP-β-CD in the aqueous phase; stationary phase: upper organic phase; mobile phase: lower aqueous phase; sample solution: 50 mg of 2-phenylpropionic acid racemate dissolved in 6 mL of the aqueous phase; flow rate: 2.0 mL/min; revolution: 800 rpm; stationary phase retention: 57%.

Fig.2. Chromatogram of HPLC analyses of PPA racemate and its preparative HSCCC fractions:(a) racemic mixture; (b) preparative HSCCC fraction containing (+)-2- phenylpropionic acid; (c) preparative HSCCC fraction containing (-)-2- phenylpropionic acid.

References

1. J.C. Ye, W.Y. Yu, G.S.Chen, Z.R. Shen, S. Zeng, Biomed. Chromatogr., 24(2010):799. 2. N. Rubio, S. Ignatova, C. Minguillón,I.A. Sutherland, J. Chromatogr. A, 1216(2009)8505. 3. S.Q. Tong, Y.-X. Guan, J.Z yan, Bei Zheng, Liying Zhao, J.Chromatogr. A, 1218(2011)5434

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-18 Excellent fraction effect of HSCCC on the completed separation of amide compounds in black pepper Yongyang Jin, Dengyong Qian, Qizhen Du* Institute of Food Chemistry, Zhejiang Gongshang University, Hangzhou 3100125, China * Corresponding author. Tel./fax: +86 571 88071024 8575. E-mail address: [email protected] (Q. Du). Keywords: black pepper; amide compounds; fraction effect; HSCCC

The genus Piper has been received considerable attention in recent years because the peppers have been used as a spice and also as a folk medicine. Black pepper, the fruit of Piper nigrum L. is widely used as an anodyne and a treatment for stomach disease in China. A lot of works on Piper species showed that the major bioactive components are amides. The preparative separation and purification of amides from plant materials by conventional methods is tedious and usually requires multiple chromatography steps, such as column chromatography (CC) and thin-layer chromatography (TLC). It is very difficult to obtain high pure amides using traditional chromatography since amides can easily suffer from peak tailing and poor efficiency on silica-based columns. Counter-current chromatography (CCC) has been used for the separations of valuable natural products due to no adsorptive sample loss and deactivation, tailing of solute peaks, contamination and etc. Thus, it is a best way to fractionate crude plant extract for separation and preparation of the whole components in the extract. In the present study, we achieved an excellent fractionation of amides in the crude extract of the fruit of Piper nigrum L., and obtained 12 amide compounds by advanced separations of the amide fractions from HSCCC fractionation. HSCCC separation employed a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:1.5:1, v/v). The upper phase was used as stationary phase while the lower phase as mobile phase. The effluent was collected by a fraction collector. The high-speed countercurrent chromatograph used in the present study was constructed at the Institute of Food and Biological Engineering, Zhejiang Gongshang University, Hangzhou, China. The apparatus was equipped with a 1200-ml column with 6-layer coils made of a 5.0 mm i.d. Teflon tubing. For the separation a K-1800 Wellchrom preparative HPLC pump (Knauer, Germany), a 100 ml sample loop made of 3 mm i.d. Teflon tubing, and a B-684 collector (Büchi, Switzerland) with 15-ml tube racks was used. The separation procedure began from the filling the column with the stationary phase. Then the apparatus was rotated at 1000 rpm and the sample solution (3.5 g crude amide components in 100 ml mobile phase) was injected into the CCC system through the Teflon sample loop with the mobile phase at a flow rate of 5.0 ml/min. The HSCCC separation of crude amide components from black pepper was showed in Fig. 1. The separation afford to 7 fractions: FrA to FrG. Concentration and dryness in vacuum of FrD, FrE and FrF resulted in compounds 10 (135 mg), 11 (85 mg) and 12 (265 mg). Preparative HPLC of FrA (100 mg) afforded compounds 1 (46 mg) and 2 (41 mg) while FrB (100 mg) gave compounds 3 (13 mg), 4 (15 mg), 5 (28 mg) and 6 (31 mg); FrC (100 mg) afforded compounds 7 (33 mg), 8 (14 mg) and 9 (17 mg); and FrG (from stationary phase) (100 mg) yielded compounds 13 (74 mg).

Fig. 1 TLC-chromatogram of 3.5 g of the crude amide compounds from black pepper. Solvent system: n-hexane-ethyl acetate-methanol-water (1:3:1.5:1, v/v), as stationary phase: upper phase, column capacity: 1200 ml, rotation speed: 1000 rpm, flow rate: 5.0 ml/min. TLC plate: Silica G 254 Aluminum, developing solvent: ethyl acetate-n-hexane (3:2, v/v); colorizing reagent: 10% sulfuric acid-ethanol solution. ESI-MS and NMR analysis indicated the 12 compounds were retrofractamide-A (1), (2E,4E)-N-isobutyl-decadienamide (2), retrofractamide-C (3), piperoleine A (4), dehydropiperoleine A (5), retrofractamide-B (6), piperoleine B (7), pipernonaline (8), pipercyclobutanamides A (9), dehydropipernonaline (10), piperine (11) , (2E,4E,12Z)-N-isobutyl-octadecatrienamide (12), and (2E,4E,14Z)-N-isobutyleicosa-2,4,14- trienamide (13). Result of the present study exhibits that HSCCC possesses excellent fraction effect for completed separation of natural products.

[1] V.S. Parmar, S.C. Jain, K.S. Bisht, R.J. Poonam, A. Jha, O.D. Tyagi, A.K. Prasad, J. Wengel, C.E. Olsen, P.M. Boll, Phytochemistry 46 (1997) 597. [2] Q. Qiao, Q. Du, H. Chen. Food Chem. 131 (2012) 1181.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-19 Multifunctional tetranortriterpenoids from Azadirachta indica: a separation challenge solved by MLCCC at both the analytical and preparative scale Hans E. Hummel*1, 2, D. F. Hein1, Y. Ma3, Y. Ito3 *1Chair of Organic Agriculture, Justus-Liebig-University Giessen, Germany. [email protected] 2Illinois Natural History Survey, Prairie Research Institute, University of Illinois, Champaign, USA 3Laboratory of Biophysical Chemistry, N.I.H., NHLB Institute, Bethesda, Md. 20892 –USA Keywords: Azadirachta indica, A. excelsa, azadirachtin, biopesticides, organic agriculture

Multilayer counter current chromatography (MLCCC) is a solid carrier free chromatographic procedure with a number of distinctive virtues essential for natural product chemists. For the separation and isolation of multifunctional tetranortriterpenoids present in Azadirachta indica A. Juss (Meliaceae) it was the method of choice. Azadirachtin (aza, Fig. 1A) and marrangin (mar, Fig. 1B) are insect antifeedants and developmental modifiers used in organic agriculture for management of pest insects. The compounds occur in seeds of the Indian (A. indica A. Juss) and the Philippinian neem tree (A. excelsa Jack) (Meliaceae), respectively. Due to irreversible adsorption of amphiphilic tetranortriterpenoids aza and mar to solid chromatographic support, attempts to purify and isolate aza and mar by classical chromatographic procedures, mainly column chromatography, HPLC and subsequent crystallization resulted in poor yields, were time consuming and quite wasteful in terms of solvent use. MLCCC provided a welcome solution to this dilemma.

A series of solvent extraction steps of crushed neem and marrango seed kernels [1-3] yielded defatted "AZT" extracts containing 6-12% of tetranortriterpenoids. This was the starting material for various purification attempts using the following semipreparative scale MLCCC machines: 1. single column MLCCC of P.C. Inc. with 315 ml coil volume; 2. double column cross-axis MLCCC instrument [4][5]; 3. triplet CPC, with 850 ml coil volume. These MLCCC runs, typically with intended overloading, resulted in aza purities between 70 and 90 % and had to be repeated by a subsequent run on the triplet CCC-1000 coil with 180 ml volume. Typical solvent systems used consisted of biphasic mixtures of hexane: EtOAc:CH3OH:H2O = 4:5:4:5 and 3:5:3:5. Monitoring was accomplished by HPLC with online UV detector operated at 216 or 254 nm and by TLC on normal phase SiO2 plates with toluene:ethylacetate: ethanol = 10:1:2 as a mobile phase. TLC spots were developed by spraying the plates with acetic acid:sulfuric acid:anisaldehyde reagent 25:1:0.1 and heating for 1 to 2 minutes at 140°C. Purities of products after 2 to 3 runs were above 97% and suitable for NMR as well as biological testing with the Epilachna varivestis and Spodoptera littoralis test systems in which the transformation of larvae to pupae and to adults could be quantitatively evaluated [6]. A special challenge was the complete separation of the closely related tetranortriterpenoids aza and mar (Fig. 1A,B) which differ only by 16 daltons. MLCCC on the triplet coil CCC-1000 achieved complete separation between the two substances. The first had a final HPLC purity of 99.6%. After several weeks in which the solvents could slowly evaporate, spontaneous crystallization of aza occurred and produced tetragonal crystals. This observation provides additional proof of the high purity achieved. Normally, aza and mar can be obtained only as amorphous precipitates.

Figure 1. Molecular structure and atomic composition of (A) azadirachtin and (B) marrangin

[1] K. Feuerhake, In: Natural pesticides from the neem tree and other tropical plants, H Schmutterer & KRS Ascher (Eds.) Proc. 2nd Int. Neem Conference (1985) 103. [2] B.H. Schneider, K. Ermel In: Natural pesticides from the neem tree and other tropical plants, H Schmutterer & KRS Ascher (Eds.) Proc. 3rd Int. Neem Conference (1987) 103. [3] H.E. Hummel, D.F. Hein, Y. Ma, Y. Ito, E. Chou, Med. Fac. Landbouww. Rijksuniv. Gent 62 (1997) 213. [4] Y. Ito, T.-Y. Zhang J. Chromatog. 449 (1988) 153. [5] Y. Ito, T.-Y. Zhang J. Chromatog. 455 (1988) 151. [6] D.F. Hein, PhD Thesis, University Giessen, Köhler Verlag, Giessen, 1999.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-20 Bioassay guided separation of bioactive components in natural products via counter-current chromatography coupled with LC/MS/MS Ruilin Hu, Peng Chen, Yuanjiang Pan* Department of Chemistry, Zhejiang University, Hangzhou 310027, China *Correspondence address: fax: +86 571 87951629. Email address: [email protected]

Keywords: Counter-current chromatography; LC/MS/MS; Extrusion methods; Drug discovery

Medicinal plants have served as an important source of drugs since ancient times. In modern pharmaceutical industries, despite the remarkable progress in synthetic organic chemistry of the twentieth century, over 25% of prescribed medicines in industrialized countries derive directly or indirectly from plants. This percentage can reach 50% when the over-the-counter (OTC) market is taken into consideration. It’s of great importance to separate the bioactive compounds in medicinal plants with high efficiency. However, the bioactive compounds may be lost during the multiple sample preparation processes when using solid-phase supporting materials. Therefore, it’s better to avoid irreversible adsorption in whole separation procedure.

Counter-current chromatography (CCC) uses two immiscible solvents without using solid matrix. The liquid stationary phase is retained by the centrifugal field. The mobile phase continuously passes through the stationary phase to achieve efficient partitions. Since CCC first developed by Ito, it has been widely considered as an efficient separation tool in natural products separation. The crude medicinal plant extract contains various compounds differing in concentrations, polarities, molecular weights and bioactivities. Rapid identification and separation of the bioactive compounds from the crude natural plant extracts inspires great interests in recent years. The extrusion CCC method (EECCC&BECCC) has been proved as an efficient fractionation method which may replace traditional fractionation procedure [1, 2]. The hydrophilic and hydrophobic components can be well separated by extrusion method. Moreover, due to the extrusion step, it saves a lot of time and solvents. On-line bioassay coupled with HPLC has been recently developed to identify the bioactive compounds in medicinal plants [3].

Due to the advantages of extrusion CCC methods and on-line bioactive assay, we combined these two methods together and applied in natural drug discovery program. The strategy of separation of the bioactive compounds is as follows: In the first step, the extrusion method was applied to rapid fractionating of the crude extract to obtain several fractions. The partition coefficients can also be obtained directly from the CCC chromatogram. In the second step, the obtained fractions were evaluated by on-line bioassay by LC/MS/MS to identify the target active compounds. In the third step, the target compounds were further isolated by CCC. The advantage of this strategy compared with traditional method used in natural drug discovery program is obvious: The bioactive compounds can be isolated in short analysis time and the minor components won’t be lost in the screen process. Two natural products Ampelopsis heterophylla and Vitis wilsonae Veitch were evaluated by this strategy. The results indicated that this strategy had great potential to be applied in natural drug discovery program.

Acknowledgement Y.P. thanks the National Science Foundation of China for Grant 21025207, 20975092.

References 1. Y. Lu, A. Berthod, R. Hu, W. Ma, Y. Pan, Anal. Chem. 2009, 81, 4048 2. Y. Lu, A. Berthod, Y. Pan, J. Chromatogr. A 1189 (2008) 10. 3. C. Sun, J. Fu, J. Chen, L. Jiang, Y. Pan, J. Sep. Sci 33 (2010) 1018.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-21 Separation and purification of quanternary protoberberine alkaloids from Corydalis saxicola Bunting by HSCCC Zhichao He1,2, Qiongxian Ye1, Junyan Wu2, Depo Yang1, Leslie Brown3, Lin Jiang1, Longping Zhu1, Dongmei Wang1* 1 School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China; 2Sun Yat-Sen memorial hospital, Guangzhou, China; 3 AECS-QuikPrep Ltd, Bridgend, S. Wales, CF31 4XZ, UK

* Dongmei Wang，Tel:+86 (020)39943042, Email: [email protected]

Keywords: Corydalis saxicola, alkaloids, HSCCC

Corydalis saxicola Bunting, a well-known Chinese herbal medicine, possesses antibacterial, antiviral, and hepatoprotective activities, etc. The active constituents were reported to be a series of tertiary and quaternary protoberberine alkaloids [1,2]. In this study, a HSCCC method for rapid separation of three main active quaternary protoberberine alkaloids was established. After partitioned by different organic solvents, the n-butanol extract of the ethanolic extract of C. saxicola was resolved in the upper layer of EA-n-BuOH-MeOH-DW-glacial HAc (1:5:0.5:5:0.1, v/v) to give a yellow participate, which was then partitioned by n-BuOH-DW (3:4, v/v) and the water layer mainly contained dehydrocheilanthifo-line (1), dehdroapocavidine (2), dehydrocavidne (3) and cavidine (4) (Fig. 1B) was selected for separation on HSCCC. Screening the K values (data not shown) of the four compounds in ten different solvent systems, n-Hex-EA-n-BuOH-EtOH-DW-ammonia (0.3:1.5:4:0.3:5:0.11, v/v, Solvent I) showed that the four target compounds might be separated on HSCCC. however, cavidine (4) was co-eluted with the other three compounds unexpectively on real HSCCC separation. In order to solve this problem, buffer solution with different pH values was used instead of ammonia in Solvent I, and cavidine could be removed when pH of buffer solution was 9.0 (Solvent II). The result in Fig.2A indicated that peak A1 contained componds 1-3, and cavidine was obtained in peak A2. The peak A1 was further separated on HSCCC by Solvent I. As shown in Fig.2B, peak B1 (3), peak B2 (2) and peak B3 (1) with purities over 95% were obtained, respectively.

Figure 1. Chromatograms of n-butanol extract of C. saxicola Bunting and pretreated sample for HSCCC. (A): Figure 2. (A) HSCCC separation of pretreated sample of n-butanol extract, (B): pretreated sample for further n-BuOH extract of C.saxicola Bunting using solvent system: separation on HSCCC; (C) peak B1 in Fig. 2B; (D) peak n-Hex-EA-n-BuOH-EtOH-W-buffer (pH=9) at 0.3：1.5：4：0.3： B2 in Fig. 2B; (E) peak B3 in Fig. 2B. Conditions: column, 5：0.11 (v/v/v/v). Stationary phase, lower phase; coil column, Phenomenex Gemini C18, 250 mm*4.6 mm ID, 5 μm; 231 mL; rotated speed, 860 rpm; flow rate, 1.5 mL/min; mobile phase, acetonitrile (A) and 20 mmol/L ammonium sample injection, 27.3 mg; detection wavelength, 280 nm; the acetate (B, adjusted to pH 4.66 by acetic acid), which was ratio retention of stationary phase was 81.4%. programmed as: 0-10 min, A: 15-20%; 10-20 min, A: (B) HSCCC separation of the eluent of peak 1 in Fig.2A using 20-22%; 20-25 min, A: 22%; 25-30 min, A: 22-24%; 30-45 ： min, 24-29%; 45-75 min, 29-90%; Flow rate, 1.0 mL/min; solvent system: n-Hex-EA-n-BuOH-EtOH-W-ammonia at 0.3 detection, 280 nm. 1.5：4：0.3：5：0.11 (v/v/v/v). Stationary phase, lower phase; coil volumn, 230 mL; rotated speed, 860 rpm; flow rate, 1.5 mL/min; sample injection, 30.0 mg; detection wavelength, 280 nm; the ratio retention of stationary phase was 82.7%.

References 1. Chemistry & Biodiversity, 2008, 5, 777-783. 2. Chemistry & Biodiversity, 2008, 5, 1335-1344.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-22 Separation and purification of four ligans from fructus arctii by high-speed counter-current chromatography Mei Yang, Xinjun Xu*, Zhisheng Xie, Jieyun Huang, Chunyan Xie School of Pharmaceutical Sciences, Sun Yat-Sen University * No. 132, East Waihuan Rd.,Guangzhou Higher Education Mega Center, 510006 Guangzhou, China e-mail address: [email protected]. fax: +86 020 39943041.

Keywords: Fructus Arctii, HSCCC, ligans, arctiin, arctigenin

Four lignans including arctiin, arctigenin, matairesinol and lappaol F were separated and purified from traditional Chinese medicine Fructus Arctii by high-speed counter-current chromatogra--phy (HSCCC). The crude extracts from Fructus Arctii were divided into two fractions by D101 macroporous resin. Fraction 1 was separated with ethyl acetate-n-butanol-water (4:0.5:5, v/v) and yielded 163.8 mg of arctiin from 250 mg of fraction 1. Fraction 2 was separated with n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v) and yielded 27 mg of arctigenin, 5 mg of matairesinol and 3 mg of lappaol F from 150 mg of fraction 2. The purities of the four compounds were 99.64%, 98.48%, 96.16% and 91.41%, respectively, as determined by HPLC. The chemical structures of these compounds were identified by MS, UV, 1H NMR and 13C NMR. Such a simple and effective method was fairly useful to prepare pure compounds as reference substances for related study on Fructus Arctii.

Figure 1 HSCCC chromatograms of fraction 1 (A) and fraction 2 (B) from Fructus Arctii. HSCCC separation conditions of A: solvent system: ethyl acetate-n-butanol-water (4:0.5:5, v/v); stationary phase: upper phase; mobile phase: lower phase; elution mode: head to tail; flow rate: 2 mL·min-1; revolution speed: 860 rpm; retention of the stationary phase: 65.2%; detection wavelength: 280 nm; sample size: 250 mg. Conditions of B: solvent system: n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v) ; stationary phase: upper phase; mobile phase: lower phase; elution mode: head to tail; flow rate: 2 mL·min-1; revolution speed: 860 rpm; retention of the stationary phase: 73.9%; detection wavelength: 280 nm; sample size: 150 mg. Peaks: I, arctiin; II, arctigenin; III, matairesinol; IV, lappaol F.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-23 Separation and Enrichment of Rare Earth Elements by Stepwise pH Gradient Countercurrent Chromatography with a Polyethylene Glycol-Na2SO4 Aqueous Two-Phase System Masami Shibukawa,* Kohei Shimizu, Shingo Saito Graduate School of Science and Technology, Saitama University * Corresponding Author. 255 Shimo-Okubo, Sakura-ku, Saitama, 338-8570, Japan. Fax: +81-48-858-3520. E-mail: [email protected].

Keywords: stepwise pH gradient CCC, pH-peak-focusing, rare earth elements, PEG-Na2SO4 aqueous two-phase system, acetylacetone.

In recent years it has been shown that high-speed countercurrent chromatography (HSCCC) can be applied for separation of various metal ions. Kitazume et al.1, 2 demonstrated that HSCCC can also be used for efficient enrichment of rare earth elements and some other metal ions using pH-peak-focusing technique. However, most of the separations of metal ions by HSCCC which have so far been reported were performed with organic solvent-water extraction systems. On the other hand, aqueous two-phase systems (ATPS) consisting of water-soluble polymers and inorganic salts have been known as an environmentally friendly extraction system and are used for separation of inorganic ions as well as organic substances.3 In this paper, we will present an HSCCC method for separation of rare earth metal ions with a polyethylene glycol-Na2SO4 ATPS. We will also show that stepwise pH gradient technique is useful not only for separation but also for enrichment of the metal ions. The experiments were performed using a type-J Hitachi Tokyo Electronics (Tokyo, Japan) high-speed counter-current chromatograph. The aqueous two-phase system used was prepared by dissolving PEG#1000 (5.4 % (w/w)), sodium sulfate (16.7 % (w/w)), and acetylacetone (50 mM) in water. The PEG-rich upper phase was used as the stationary phase, while the salt-rich lower phase was used as the mobile phase. The column was first entirely filled with the stationary phase, and then the sample solution was injected through the sample port and the mobile phases of different pH were delivered in stepwise gradient elution mode. The rare earth elements (La, Ce, Nd, Yb, Sc) were detected by means of a postcolumn reaction with Arsenazo III and the chromatograms were obtained by monitoring the absorbance at 650 nm with a spectrophotometer. Rare earth metal ions were extracted into the PEG-rich phase by complexation with acetylacetone. Stability constants of the complexes depend on the nature of the rare earth metal ions. Therefore, pH values at which the extraction takes place are also dependent on the type of the metal ions. This means that mutual separation of rare earth elements can be performed by HSCCC using a stepwise pH gradient elution. Furthermore enrichment of the individual metal ions is expected to occur at the pH borders of different mobile phases due to pH-peak-focusing. Figure 1 shows a chromatogram obtained by HSCCC with a pH stepwise gradient elution of five mobile phases. It should be noted that not only mutual separation but enrichment of the metal ions are achieved. We will also discuss the mechanism of formation of pH gradient profile in the ATPS-HSCCC and the potential of this technique for preparative scale separation. 9.0 Nd 8.0 Sc 7.0 La Ce Yb 6.0 pH

5.0

Absorbance Absorbance 4.0

pH 7.5 pH 7.1 pH 6.5 pH 5.9 pH 3.0 3.0 HEPES BES PIPES MES H SO 2 4 2.0 0 50 100 150 200 time (min) FigureFig.1 Chromatogram1. Chromatogram of rare earthof elementsselected and pHrare profileearth of elementseluate. obtained by stepwise pH gradient HSCCC eferences 1. Kitazume, E.; Higashiyama, T.; Sato, N.; Kanetomo, M.; Tajima, T.; Kobayashi, S.; Ito, Y. Anal. Chem. 1999, 71, 5515-5521. 2. Kitazume, E.; Takatsuka, T.; Ito, Y. J. Liq. Chromatogr. & Rel. Technol. 2004, 27, 437-449. 3. Shibukawa, M.; Nakayama, N.; Hayashi, T.; Shibuya, D.; Endo, Y.; Kawamura, S. Anal. Chim. Acta 2001, 427, 293-300.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-24 Application of High-speed Countercurrent Chromatography, TLC and nano-LC-MS/MS in Lipidomics

Majiaojiao1, Lili Niu1, Tanxi Cai1, Yoichiro Ito2 and Fuquan Yang1* 1Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

Lipids constitute the largest subset, including tens of thousands of distinct lipid molecular species existing in the cells and tissues. Lipids play multiple and critical roles in cellular functions, such as composing the membrane bilayer, providing an appropriate hydrophobic environment for membrane proteins and their interactions, and participating in cell growth, multiplication, and death. Simultaneously, some lipids are messengers in cell signaling transduction processes and can be utilized as biomarkers of some diseases.

Lipidomics, a dominant part of metabolomics, is the detailed analysis and global characterization, both spatial and temporal, of the structure and function of lipids (the lipidome) within a living system. Comparing the lipidome of healthy versus diseased states can provide information helpful in correlating the role of lipids in various diseases, such as cancer, atherosclerosis, and chronic inflammation. Additionally, a large variety of lipid species comprise cellular membranes, and investigating their interactions with membrane-associating proteins/enzymes can provide insight into such areas as drug/inhibitor interactions.

Lipids are divided into eight categories, namely fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides, and most lipids are of hydrophobicity. As a result of the complexity and diversity of lipids, efficient separation and accurate identification and comprehensive classification and are required for lipidomics. Chromatography and mass spectrometry (MS) have become the major tools for lipidomics. Various separation technologies, such as thin-layer chromatography (TLC), gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE), are well accepted and open up new applications in lipidomics research through coupling with MS. In this study, HSCCC is first used for the fractionation of lipids from human sera, hela cells and Caenorhabditis elegans, followed by TLC and HPLC-ESI-MS/MS analysis. Four two-phase solvent systems are tested for HSCCC separation in this study.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-25 Isolation of two new diterpenoids from clerodendrum kaichianum hsu. by high-speed counter-current chromatography using stepwise elution Ming-feng Xua, Qi-zhen Dub,*, Lian-qing Shenb, Hui-zhong Wanga aCollege of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China bCollege of Food Science and Biotechnology Engineering, Zhejiang Gongshang University, Hangzhou, 310035, China *Correspondence: Prof. Qi-zhen Du, E-mail: [email protected] Fax: +86-571-88071024-8575 Keywords: C. kaichianum Hsu; Diterpenoid; High-speed counter-current chromatography; stepwise elution;

Clerodendrum is a genus of about 400 species in the family Verbenaceae, which mainly grow in the tropical and warm temperate zones including Africa and southern Asia. A few of species are distributed in America, northern Australia and eastern Asia. Plants of the genus Clerodendrum are well known for treatment of different diseases, such as asthma, pyreticosis, catarrhal affections of the lungs fever, inflammation and skin diseases as well as asthma. In China, the leaves of C. kaichianum Hsu are used as a traditional medicine against hypertension. Separation of abietane-type diterpenoids from C. kaichianum Hsu by high-speed counter-current chromatography using stepwise elution was studied in this paper. Since the different retention of stationary phase of different system composition, the change of speed was studied in order to maintain the retention of stationary phase with different system. After the flow rate of the HSCCC system was adjusted to give the same retention of stationary phase as that of the first system, these optimized parameters were successfully change to the second system for the separation and purification of four compounds from C. Kaichianum. The HSCCC was performed on a HSCCC instrument equipped with a 1100-mL column, using the upper phase of light petroleum–ethyl acetate–methanol–water (PEMW) (5:5:3:6, v/v/v/v) as stationary phase, and the lower phase of PEMW as mobile phase, later changed to (PEMW) (5:5:5:5). Using the optimized conditions, two new and two known diterpenoids were obstained, 2 g of crude extract was separated to yield 19.3 mg of A, 25.9 mg of B, 12.4 mg of C and 18.6 mg of D with purities 81.2%, 85.7%, 95.3% and 96.2%, respectively. Their structures were identified by spectroscopic methods and ESI-MS.

Figure 1. Separation of four diterpenoidss from the stems of C. kaichianum Hsu by HSCCC.

This is the first report that abietane diterpenoids from the stems of C. kaichianum Hsu were successfully isolated and purified by the stepwise counter-current chromatography method, which demonstrated that stepwise HSCCC is a fast, effective and powerful technique for the isolation and purification of diterpenoids from natural herbs. References

1. Editorial board of The Flora of China The Flora of China (Science Press, Beijing) 1994. 65, pp. 150-151. 2. H.H. Nan, J. Wu, and S. Zhang, Pharmazie 2005,60, 798. 3. S.S. Liu, T.Z. Zhou, S.W. Zhang, and L.J. Xuan, Helv. Chim. Acta 2009, 92, 1070. 4. R. Ravikumar, A.J. Lakshmanan, and S. Ravi, J. Asian Nat. Prod. Res. 2008, 10, 652. 5. T. Kanchanapoom, P. Chumsri, R. Kasai, H. Otsuka, and K. Yamasaki, J. Asian Nat. Prod. Res. 2008, 7, 269. 6. K.H. Kim , S.G.. Kim, M.Y. Jung, I.H. Ham, and W.K. Whang, Arch. Pharm. Res. 2009, 32, 7.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-26 Preparative isolation of Oolong tea polyphenols by high-speed countercurrent chromatography Kunbo Wang, Fang Liu, Zhonghua Liu*, Jian-an Huang, Yong Lin, Yushun Gong National Research Center of Engineering & Technology for Utilization of Botanical Functional Ingredients, Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha, Hunan, 410128, China. * Correspondence address, National Research Center of Engineering & Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Furong district, Changsha, Hunan 410128, People’s Republic of China, email: [email protected] Keywords: High-speed countercurrent chromatography; Oolong tea; catechins.

Abstract: The characteristic feature of oolong tea components is that it contains numerous kinds of polymerized-polyphenols, which are derived from tea catechins by polyphenol oxidases or by heating process, which is called semifermented process. Oolong tea polymerized-polyphenols, EGCG, gallocatechin gallate and caffeine, which are the major compounds of oolong tea extract, were examined (1). Isolation of tea polyphenols or catechins has been based on chromatography on (semi-) preparative HPLC and sephadex LH-20 (2,3). The procedures are times consuming and tedious. Thereafter, it is ideal for the separation of tea polyphenols or catechins by HSCCC (4-6). High-speed countercurrent chromatography (HSCCC) has been applied for the separation of oolong tea polyphenols. The HSCCC run was carried out with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25, v/v) by eluting the lower aqueous phase at 2 ml/min at 700 rpm. The results indicated that catechins including epigallocatechin gallate, gallocatechin gallate and epicatechin gallate were isolated from the aqueous extract of oolong tea (Fig.1). Fujian Tieguanyin, Dahongpao, Guangdong Fenghuang Dancong and Taiwanese oolong have similar HSCCC profiles.

Fig.1 Preparative separation of oolong tea polyphenols by HSCCC

Solvent system: hexane–ethyl acetate–methanol–water–acetic acid (1/5/1/5/0.25, v/v); mobile phase: lower aqueous phase; low-rate: 2 ml/min; column volume: 243.0 ml; sample size: 40 mg; retention of stationary phase: 66.3%; 1,2,4: unknown; 3: EGCG; 5: GCG; 6: ECG.

References

1. M. Nakai, Y. Fukui, S. Asami, Y. Toyoda-Ono, T. Iwashita, H. Shibata H et al, J. Agric. Food Chem. 538 (2005) 4593. 2. R. Amarowicz, F. Shahidi, Food Res. Intern. 29 (1996) 71. 3. A. L. Davis, Y. Cai, A. P. Davies, J. R. Lewis, Magn. Reson. Chem. 34(1996) 887. 4. A. Degenhardt, U. H. Engelhardt, C. Lakenbrink, P. Winterhalter, J. Agric. Food Chem. 48 (2000) 3425 5. T. Y. Zhang, X. L. Cao, X. Han, J. Liq. Chrom. Rel. Technol. 26 (2003) 1565. 6. K. B. Wang, Z. H. Liu, J.A. Huang, X. R. Dong, L. B. Song, Y. Pan et al, J. Chromatogr. B 861 (2008) 282.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-27 Generation of knock-out extracts by countercurrent chemical subtraction René F. Ramos, Guido F. Pauli, and Shao-Nong Chen1,* 1UIC/NIH Botanical Center, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, 833 S. Wood St., Chicago, IL 60612, USA * [email protected] fax +1 312 996 7107 Keywords: Chemical subtraction, Humulus lupulus, xanthohumol, isoxanthohumol, 8-prenylnaringenin

Unlike in mathematics, where subtraction is a basic operation, subtraction in chemistry is a non-trivial task for any separation technology. In particular, solid-phase based chromatography suffers from unavoidable absorption, making it essentially unsuitable for studies involving bioactivity and quantitation. Suitable methods for chemical subtraction are in high demand for studies at the chemistry-biology interface, such as in natural products research.

Recently, the generation of “knock-out (KO) extracts” has been reported, using affinity chromatography with monoclonal antibodies for the removal of single phytoconstituents from plant extracts. Another approach, termed chemical subtraction, has been pioneered in our laboratory and uses countercurrent chromatography (CCC) to deplete target(s) from a complex mixture, following the equation: extract (minuend) – target compound (subtrahend) = knock-out extract (difference). Chemical subtraction enables further chemical and biological characterization of both the otherwise intact KO extract as well as the residually complex subtrahend(s).

The concept of chemical subtraction by CCC has now been further developed and applied to the following bioactive prenylated phenols contained in the dietary supplement, hops: xanthohumol [XH], isoxanthohumol [IX], and 8-prenylnaringenin [8-PN]. The approach uses HSCCC and HEMWat 0 as the initial solvent system. Reflecting their different abundance in the extract and differences in CCC selectivity, the subtracted XH (33%), IX (3%), and 8-PN (0.35%) had qHNMR purities of 90, 54, and 12% w/w, respectively. The low 8-PN purity also reflects its co-elution with XH. Thus, a second CCC subtraction was performed using HEMWat -3 and orthogonal HterAcWat -3 systems. All HSCCC fractions other than those of the targets were recombined to provide the KO extracts.

Quantitative LC-MS, qHNMR and UHPLC profiles will be presented for four hops KO extracts: XH-KO, IX-KO, 8-PN-KO and XH/IX/8-PN-KO.

The method developed for hops exemplifies the potential of the chemical subtraction concept for K-based targeted analysis, and opens new opportunities in research on bioactive natural products.

Figure 1. Structures of prenylphenols from Humulus lupulus which were chemically subtracted from crude extracts.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-28 Rapid Separation of Polyphenols from Green Tea by HSCCC and Characterization the interaction between EGCG and Protein by ACE WANG Wei*, LE Sheng-feng, ZHAO Xin-ying, WANG Tan, ZHANG Jing-hua Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China *Correspondence: Block B 4/F, Incubation Building, No.7, The Middle of Fengxian road, Haidian District, Beijing City; fax: 010-58717638; e-mail: [email protected]

Keywords: Green tea, EGCG, High-speed countercurrent chromatography, Affinity capillary electrophoresis, Bovine serum albumin

Tea, one of the most widely consumed beverages throughout the world, contains a wide range of polyphenols, such as catechins, flavonoid glycosides and phenolic acids. Among of them epigallocatechin gallate (EGCG) is regarded as the most important of the tea catechins. It has been recognized that the catechins possess many biological activities and pharmacological actions including antioxidant, anti-allergy, anti-virus, reduced risk of cardio-vascular injury et al.

In order to separate the main polyphenols from green tea, high-speed countercurrent chromatography (HSCCC) was applied. Green tea was extracted with 80% ethanol (v/v) by ultrasonic-assisted. After various parameters of HSCCC were optimized preliminarily, the crude extract of the EtOAc part of green tea was separated with a two-phase solvent system composed of EtOAc-Ethanol-water (10:1:10, v/v/v). The coiled column was first entirely filled with the organic stationary phase, and the Model TBE-20A analytical HSCCC was rotated at 1800 rpm, while the aqueous mobile phase was pumped into the column at a flow-rate of 1.0 mL/min (Figure 1). The purity and identity of EGCG was confirmed by 1H NMR and HPLC-MS.

Figure 1 Chromatograms of crude polyphenol Figure 2 Electropherograms of EGCG and extract from green tea obtained by HSCCC BSA based on ACE 1 mM EGCG and BSA 0 μM (A). BSA 3 μM (B). BSA 5 μM. (C) BSA 10 μM. (D) BSA 15 μM (E). The interaction between EGCG and protein (Bovine Serum Albumin, BSA) was determined by affinity capillary electrophoresis (ACE). The separation was performed at 40/48.5 cm capillary with 10 kV voltage and 214 nm detection. The electrophoretic mobility changes of EGCG were measured with various concentrations of BSA added to the borax running buffer, pH 8.7 (Figure 2). The binding constant was calculated from the electrophoretic mobility change to be 1.28×105 L/mol.

The analytical HSCCC is a quick tool for isolation of individual catechins with high purity. And the binding constant between EGCG and BSA determined by ACE is in good agreement with that reported in the literature. The present work offers a good quick method for study on affinity interactions of drug-protein from crude extract, which can be generally applied to a wide of screening active ingredients.

References

Cao X L, Tian Y, Zhang T Y, et al. J. Liq. Chromatogr. Related. Technol. 2001, 24, 1723-1732. Chu Y H, Avila L Z, Gao J M, et al. Acc. Chem. Res. 1995, 28(11), 461-468.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-29 Online isolation and purification of four phthalide compounds from Rhizome chuanxiong using counter-current chromatography coupled with semi preparative high performance liquid chromatography Yun Wei *, Wenwen Huang *State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China. Tel & Fax: 0086 10 64442928. E-mail: [email protected] Keywords: Phthalide Compounds, Counter-current Chromatography, Semi Preparative High Performance Liquid Chromatography ABSTRACT Rhizome chuanxiong is one of the most widely used traditional herbal medicines. There are three types of pharmacologically active components in Rhizome chuanxiong, namely phenolic acid, alkaloid, and phthalide. Furthermore, the phthalide compounds including senkyunolide A (1), levistolide A (2), Z-ligustilide (3) and 3-butylidenephthalide (4), have been reported as the biologically active compounds contributing to the therapeutic effects of this medicinal herb[1]. In this study, a counter-current chromatography coupled with semi preparative high performance liquid chromatography through a six-way valve has been used to achieve online isolation and purification of the four components simultaneously. Through the solvent system selection experiment, Compound (3) and (4) were eluted in one peak and could not be separated, which is common in countercurrent chromatography separation [2-3]. So a six-way valve was used to cut the peak of the mixture and injected to the semi preparative high performance liquid chromatography directly. By using a solvent system of n-hexane-ethylacetate-methanol-water-acetonitrile ( 8:2:5:5:5, v/v ) , 3.6 mg (1) (94.43%), 3.0 mg (2) (95.31%) were obtained, and the 8 ml of (3)+(4) were transferred and injected for further separation by the semi preparative high performance liquid chromatography. 5.6 mg (3) (97.5%) and 4.8 mg (4) (99.3%) were obtained. The identification of the four compounds were performed by ESI-MS, 1H-NMR and 13C-NMR spectra.

Figure 1 Scheme of the CCC-Pre-HPLC system Figure 2. Chromatogram of the crude extract from 110 100 3 Rhizome chuanxiong by HSCCC. solvent 90

80 4 system :n-hexane-ethylacetate-methanol-water-a 70 60 cetonitrile ( 8:2:5:5:5, v/v ); sample: 100 mg crude

mAu 50 40 extract dissolved in 0.5 ml lower phase and 0.5 ml 30 20 upper phase; 1 = senkyunolide A; 2 = levistolide A; 10 0 3=Z-ligustilide; 4=3-butylidenephthalide .

0 5 10 15 20 25 30 35 40 45 50 time(min)

Figure 3. Chromatogram of mixture of (3)+(4) by Pre-HPLC. reversed phase C18 column (250×20mm.I.D., 5µm,YMC-Pack ODS-A). Mobile phase:

MeOH/H2O (65:35, v/v). Flow rate: 8ml/min. 3= Z-ligustilide; 4 = 3-butylidenephthalide. References (1) Yan, R.; Li, S.L.; Chung, H.S.; Tam, Y.K.; Lin, G. J. Pharm. Biomed. Anal.2005, 37, 87-95 (2) Wei, Y.; Xie, Q.Q.; Dong, W.T.; Ito, Y. J. Chromatogr. A. 2009,1216, 4313–4318. (3) Beer, D.; Jerz, G.; Joubert, E.; Wray, V.; Winterhalter, P. J. Chromatogr. A. 2009,1216, 4282–4289.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-30 Separation of 11-cis-retinal from the retinal isomers by flash countercurrent chromatography 1Minfei He, 1Wenkai Du, 1Qingbao Du, 1Yun Zhang, 1Bo Li, 2Changqian Ke, 2Yang Ye*, 1Qizhen Du* 1Institute of Food Chemistry, Zhejiang Gongshang University, Hangzhou 3100125, China 2State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China Corresponding author. Tel.: +86-215-0806726; fax: +86-215-0806726. E-mail address: [email protected] (Y. Ye). Tel.: +86 571 88071024; fax: +86 571 88823509. E-mail address: [email protected] (Q. Du); Keywords: 11-cis-retinal; isomerization; isomerization; separation; FCCC 11-cis-retinal is a key substance for visual pigment research since it is the chromophore of rhodopsin. The instability of 11-cis-retinal causes lack of commercial supply. The preparation of 11-cis-retinal has been achieving by an isomerization reaction of all-trans-retinal. But the separation of 11-cis-retinal from the reaction products was so easy, especially for a mount with milligram scale. Column chromatography on alumina, thin-layer chromatography (TLC) were used to the separation at early. The methods suffered from several disadvantages: (i) only a limited amount can be applied to a plate, (ii) the separation of the 11-cis and 13-cis isomers is poor, (iii) the material is particularly prone to degradation while adsorbed on the stationary phase. Following, high-performance liquid chromato-graphy (HPLC) has been applying to the separation of retinal isomers, but the loading amount was lower than 1 mg of the products of the isomerization reaction on a preparative column (Supelcosil LC-Si, 250 x 21.2 mm, 5 µm). In the present paper, we describe a HSCCC separation of the products of the isomerization reaction with a mount of sample load more than 60 milligram.

O 13 9 11 all tran-retinal The solution of all-trans-retinal is irradiated in a plate of 96 cells placed 15 cm from a 1000 W high-pressure mercury arc lamp (Thorn Lighting, Type MED) with another as a stopper. The light is passed through a heat-absorbing glass filter. The light absorbed by the retinal is primarily the 436 nm mercury line. In each irradiation, 96 x 0.3 ml of a solution containing 4.0 mg/ml all trans-retinal in acetonitrile is placed in the cells. The reaction solution was cooled to 3°C and an irradiation was performed for 2 min. After the irradiation finished, the solution of the isomerization reaction was directly subjected to FCCC separation.

The preparative FCCC experiment was performed on a HSCCC instrument with 1200 column made of 5.0 i.d. bore tubing. The sample solution was prepared by adding n-hexane into 50 ml of the isomerization solution to saturation of n-hexane (40 ml). The mobile phase was pumped at a flow rate of 25.0 ml/min when the apparatus was rotated at 1000 rpm. The effluent was monitored by a UV detector at 236 nm and collected by a fraction collector with 25 ml/tube. The separation chromatogram was showed in Fig. 1.

Fig. 1 Chromatogram of FCCC separation of 50 ml of isomerization solution (4 mg/ml of all trans-retinal). Column capacity: 1200 ml, solvent system: n-hexane-acetonitrile (3:1), mobile phase: upper phase, flow rate: 25 ml/min, rotation speed of column: 1000 rpm. I: 13-cis-retinal, II: 11-cis-retinal, III: 9-cis-retinal, IV: all-trans-retinal. In each injection run, the separation yielded 63 mg of 11-cis-retinal, 24 mg of 13-cis-retinal and 26 mg of 9-cis-retinal with a purity of 97%. The method can meet the preparation for the research of bioactivity of 11-cis-retinal, 9-cis-retinal and 13-cis-retinal.

[1] Knowles, A.; Priestley, A. The preparation of 11-cis-retinal. Vision Res. 18 (1978) 115-116. [2] Qiao, Q.; Du, Q. Preparation of the monomers of gingerols and 6-shogaol by flash high speed counter-current chromatography. J. Chromatogr. A, 1218 (2011) 6187- 6190.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-31 Nmr-guided countercurrent purification and quantification of green tea catechins Feng Qiu1, Samuel Pro2, Guido F. Pauli1, J. Brent Friesen1,3,*

1Department of Medicinal Chemistry & Institute of Tuberculosis Research, University of Illinois, College of Pharmacy, Chicago, IL 60612, USA; 2Wrightwood Technologies Inc., Chicago, IL 60660; 3Physical Sciences Dept., Rosary College of Arts and Sciences, Dominican University, River Forest, IL 60305, USA. *[email protected] fax +1 (708) 488-5039 Keywords: qNMR, Camellia sinensis, catechins, countercurrent separation

Nuclear Magnetic Resonance (NMR) was used to measure K values, identify analytes, and determine product purity for the high-speed countercurrent chromatography (HSCCC) isolation of major and minor catechins from an ethanolic extract of green tea (Camellia sinensis). Four possible solvent systems (SSs) for the separation of green tea catechins were identified by the Generally Useful Estimation of Solvent Systems (GUESS) method. The K values of catechin (C), epicatechin (EC), epigallocatechin gallate (EGCG), epigallocatechin (EGC) and epicatechin gallate (ECG) were measured by quantitative 1H NMR (qHNMR) analysis of the upper and lower phases of a shake-flask experiment. At the same time, the presence of other phytoconstituents such as caffeine and gallocatechin gallate could also be observed in the NMR spectra. Three rounds of NMR-guided SS selection were performed to choose appropriate formulations from hexane: ethyl acetate: methanol: water, methyl tert-butylether: acetonitrile: water, ethyl acetate: 1-butanol: water, and chloroform: methanol: water, for primary and secondary fractionation steps with orthogonal characteristics. Analytical and preparative scale HSCCC separations were performed on a prototype CherryOne CCC control system which allowed for direct monitoring of real-time stationary phase volume retention, real-time K values, and real-time eluent composition. The CherryOne also features the remote operation and analysis of the separation process. Each CCC fraction was weighed in order to create a mass chromatogram of the entire separation. NMR of analysis of combined fractions from the primary fractionation step facilitated the identification of analytes and informed the selection of the appropriate secondary fraction steps employing orthogonal SS in which purified green tea catechins were obtained and measured for purity with qHNMR techniques. NMR-guided isolation and gravimetric quantification of target analytes avoids several challenges associated with HPLC-guided isolation and analysis such as: (a) the need to develop a chromatographic method for HPLC; (b) acquisition and characterization of identical reference standards required to identify and quantitate HPLC peaks; (c) variation of sensitivity inherent in different detection methods that influences quantitation; and (d) the transparency of certain impurities in HPLC detection.

Figure 1. NMR profiles of the upper and lower phases of a shake-flask experiment with Green Tea extract.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-32 Separation of bioactive components from Gynura divaricata(L.) DC by high-speed counter-current chromatography Lu Yin, Xueli Cao*, Jing Xu, Chao Cheng, Hairun Pei

Beijing Key Lab of Plant Resources Research and Development, School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing 100048, China * E-mail: [email protected]; Fax: 00-86-10-68984898 Keywords: Gynura divaricata(L.) DC.; High-speed countercurrent chromatography (HSCCC); bioactive components

Gynura divaricata (L.) DC is a traditional medicinal and edible plant in China. With its authentication as a new resources food by China State food & Drug Administration in 2010, a variety of its biological effects and health functions draw much more public attention, especially its hypoglycemic effect. However, the material basis of this effect is not very clear at this moment, more intensive and systematic research is necessary. In order to investigate potential bioactive components from Gynura divaricata(L.) DC., the 65% ethanol extract of the stem and leaf of Gynura divaricata(L.) DC. was fractioned by extraction with petroleum ether, ethyl acetate and n-butanol successively. According to the results of active assay, the ethyl acetate fraction was subjected to further separation. In this paper, four phenolic acids and one flavonoid were separated and purified from Gynura divaricata(L.) DC. using high-speed counter-current chromatography (HSCCC). The solvent system was selected by analytic HSCCC with EECCC mode based on ARIZONA system. Firstly, the solvent system composed of hexane: MtBE: methanol: 0.1% TFA aqueous (1:9:1:9, v/v) was employed Figure 1. The structures of compounds purified from for preparative separation. Four pure compounds were Gynura divaricata(L.) DC. obtained within 60min from 300 mg ethyl acetate extract, including 8.8 mg of 4,5-dicaffeoylquinic acid (Isochlorogenic acid C, 2), 35.1 mg of 3,4-dicaffeoylquinic acid (Isochlorogenic acid B, 3), 12.6 mg of 3,5-dicaffeoylquinic acid (Isochlorogenic acid A, 4) and 10.6 mg of kaempferol-3-O-β-D-glucopyranoside (Astragalin, 5). Then, 22.4 mg of 3-O-caffeoylquinic acid(Chlorogenic acid, 1) were obtained from 300mg ethyl acetate extract by HSCCC using the solvent system composed of MtBE: methanol: 0.1% TFA aqueous (10:1:9, v/v). The purities of the separated compounds were all over 98% determined by HPLC. The chemical structures were confirmed by 1H-NMR, 13C-NMR and ESI-MS.

References

1. Chunpeng Wan, Yanying Yu, Shouran Zhou, Shuge Tian, and Shuwen Cao. Isolation and identification of phenolic compounds from Gynura divaricata leaves. Pharmacognosy Magazine, 2011, 7(26): 101–108.

2. Min Jie, Chunpeng Wan, Yanying Yu, Shuwen Cao. Separation of flavonoids from crude extract of Gynura divaricata by macroporous resin. Asian Journal of Chemistry, 2011, 23(9): 3964-3968.

This project is financially supported by National Natural Science Foundation of China and Beijing Natural Science Foundation

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-33 Combination of High-Speed Counter-Current Chromatography and Preparative HPLC to Separation of Flavonoid Glycosides and Caffeoylquinic Acid Derivatives from Leaves of Lonicera japonica Thunb. Daijie Wang1, 2, Jinhua Du1*, YunLiang Lin2, Shengbo Li3, Xiao Wang2 1 College of Food Science and Engineering, Shandong Agricultural University, 61 Daizong Street, Taian 271018, P. R. China; 2 Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, P. R.China; 3 Shandong Yate Eco-tech Co. LTD, Linyi 266071, China *Correspondence: Jinhua Du. E-mail: [email protected], Tel & Fax: +86 538 8249157 Keywords: Lonicera japonica Thunb.; flavonoid glycosides; caffeoylquinic acid derivatives; high-speed counter-current chromatography; preparative HPLC

ABSTRACT Lonicera japonica Thunb. (Caprifoliaceae plants) is one of the most common traditional Chinese medicines and used for treating various diseases, including arthritis, diabetes mellitus, fever and infections. The leaves of L. Japonica is not fully research, it is necessary to develope an efficient method to separate and purify active compounds. The scavenging ability on ROS free radical such as O2‾• and •OH of the n-butanol extract from leaves of L. Japonica in vitro were determined by flow injection chemiluminescence. When the concentration was 2.0 mg/mL, ‾ the clearance rate of O2 • was 97.03%. And the clearance rate of •OH was 95.57% at the concentration of 4.0 mg/mL. n-butanol extracts have good ability of natural antioxidant and possess a certain value of applications. Then, HSCCC together with preparative HPLC were used for the separation of flavonoid glycosides and caffeoylquinic acid derivatives. 1.0 g of the n-butanol extract was firstly isolated by HSCCC using a two-phase solvent system composed of methyl tert-butyl ether-n-butanol-acetonitrile-water (0.5% acetic acid) (2: 2: 1: 5, v/v), yielding five fractions F1, F2, F3, F4, F5 (collected from the column after the separation). F1 is the mixture of chlorogenic acid, lonicerin and rutin. This mixture was separated by preparatve HPLC successfully. F2 and F3 were rhoifolin and luteoloside. F4 (mixed with 3,4-O-dicaffeoylquinic acid and hyperoside) and F5 (mixed with 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid) were separated by preparatve HPLC successfully. Combination these two methods, 10.9 mg of chlorogenic acid (1), 30.7 mg of lonicerin (2), 16.7 mg of rutin (3), 21.5 mg of rhoifolin (4), 32.0 mg of luteoloside (5), 20.3 mg of 3,4-O-dicaffeoylquinic acid (6), 18.2 mg of hyperoside (7), 24.7 mg of 3,5-O-dicaffeoylquinic acid (8) and 26.1 mg of 4,5-O-dicaffeoylquinic acid (9) were yielded, with the purities of 99.5%, 98.7%, 99.3%, 94.3%, 96.1%, 97.1%, 97.4%, 96.9% and 97.8%, respectively, as determined by HPLC. The structures of these compounds were identified by ESI-MS, 1H NMR and 13C NMR. Being a kind of support-free all-liquid partition chromatography, HSCCC can eliminate irreversible adsorption of sample on the solid support. It is an excellent enrich method for separation similar polity compounds.

µ µ µ µµF µµF (254nm) µ µ µ 1 µµF µµF 3 4 µ 2 µ µ Absorbance µ µ

µ µ µ µ µ µ µ µ µ µµt/min µµt /h Fig. 1. The HPLC analysis of the n-butanol extractFig. 2. HSCCC chromatogram of n-butanol extract

R3O 6 OH 2 R1=H R2=OGlc(2-1)Rha R3=OH 5 1 R1=caffeoyl R2=H R3=H R2O 4 CO H 3 R1=OGlc(6-1)Rha R2=OH R3=OH 2 2 3 1 6 R =caffeoyl R =caffeoyl R =H R O 1 2 3 2 R 4 R =H R =OGlc(2-1)Rha R =H 3 1 2 3 OR1 OH 6' 8' 9' 5' COO 8 R =caffeoyl R =H R =caffeoyl 5 R1=H R2=OGlc R3=OH 1 2 3 R1 4' 1' Caffeoyl= HO 7 R1=OGal R2=OH R3=OH 7' OH O 9 R1=H R2=caffeoyl R3=caffeoyl 3' 2' HO Fig. 3. The structures of isolated nine compounds

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-34 Preparative separation of conjugated linoleic acids (CLA) from camellia oleifera abel using ph-zone-refining counter-current chromatography Guanglei Song, Jingbo Wang, Yanbo Wang, Qizhen Du*

Institute of Food and Biological Engineering, Zhejiang Gongshang University, 149 Jiaogong Road, Hangzhou, Zhejiang 310035, China * Corresponding author. Tel.: +86 571 88071024; fax: +86 571 88823509. E-mail address: [email protected] (Q. Du).

Keywords: Camellia oleifera Abel, Conjugated linoleic acid, pH-zone-refining high-speed counter-current chromatography, Separation

The paper concentrates especially on the separation of three Conjugated linoleic acid (CLA) isomers, 12-hydroperoxy-8-trans,10-trans-octadecadienoate,9-hydroperoxy-10-trans,12-cis-octadecadienoate and 13-hydroperoxy-9-cis,11-trans-octadecadienoate, from Lactic Acid Bacteria fermented Camellia oleifera Abel cake. The elution sequence of the isomeric CLA, the mixing zone and mechanism of separation are discussed. The separation (Figure1) of 465.9mg of the crude sample yielded three isomeric compounds: 151.3mg12-hydroperoxy-8-trans,10-trans-octadecadienoate,84.1mg 9-hydroperoxy-10-trans,12-cis-octadecadienoate and 79.7 mg 13-hydroperoxy-9-cis,11-trans-octadecadienoate at a high purity of over 98%, 94% and 96%, respectively.

Figure 1. pH-zone-refining HSCCC separation of C. oleifera Abel extract. Solvent system: heptane/acetonitrile/acetic acid/methanol (4:4:1:1, v/v), 20mM NH4OH in the upper organic stationary phase and 10mM HCl in the lower aqueous phase; sample size: 465.9 mg (A) and 1.216 g (B); flow-rate: 2 mL/min; detection: 233 nm; revolution speed: 850 rpm; retention of stationary phase: 39.2% (A) and 34.9% (B).

References

1. Pajunen, T. I., Koskela, H., Hase, T., Hopia, A. Chem. Phys. Lipids. 2008, 154, 105 2. Tong, S., Yan, J., Guan, Y. X. J. Chromatogr. A 2008,1212, 48 3. Weisz, A., Idina, A., Ben-Ari, J., Karni, M., Mandelbaum, A., Ito, Y. J. Chromatogr. A 2007, 1151, 82 4. Fritsche, J., Fritsche, S., Solomon, M. B., Mossoba, M. M., Yurawecz, M. P., Morehouse, K., Ku, Y. Eur. J. Lipid Sci. Technol. 2000, 102, 667

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-35 From medicinal plant to compound: isolation of shikimic acid from Illicium verum by hyphenated expanded bed adsorption chromatography and counter-current chromatography Ping Hu1, Man Liu1, Min Zhang2, *, Yingxi Hua1, Hongyang Zhang1, Yuerong Wang1, Guoan Luo2,3 ** 1 School of Chemistry and Molecular Engineering; 2 School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China 3 Department of Chemistry, Tsinghua University, Beijing 10086, China * Corresponding authors: G.A. Luo, e-mail: [email protected];M. Zhang, e-mail: [email protected].

Keywords: shikimic acid; Illicium verum; counter-current chromatography; expanded bed adsorption chromatography; hyphenation

Abstract

Counter-current chromatography (CCC) has been developed for over 40 years and been applied in various fields. Those applications, particularly in natural products, have shown great advantages in high recovery and short processing time [1]. Natural compounds, which researchers are interested in, are usually present in medicinal plants at very low levels, which make the isolation process tedious and expensive. However, before the separation by CCC, the crude materials need long processing procedure such as extraction, filtration, concentration and preliminary separation to prepare crude samples for CCC. How to achieve the target compounds from medicinal plants in a fast, accurate and comprehensive way will be the main challenge for researchers. Expanded bed adsorption chromatography (EBAC) is a unique technique which integrated clarification, concentration and purification into one operation unit [2]. The new operation unit eliminates the steps of centrifugation and filtration, saves operating time, improves the yield of target compounds and reduces the cost of purification.

A new method for deriving drug discovery leads from medicinal plants is presented in this paper. This method involves: 1) hyphenating EBAC to CCC via an interface unit, 2) in the 1st dimension, preliminary separation of targets by integrated EBAC, 3) CCC solvent system selection and matching, 4) in the 2nd dimension, fine separation of targets by CCC.

This method was used in the following case study. In the extraction step, a certain amount (8.0 g) of Illicium verum was put into the continuous extraction column with an ultrasonic assisted extraction device outside. Ion exchange resins (D293) were packed into the expanded bed chromatography column. Target compound-shikimic acid was extracted in the continuous extraction column and simultaneously the target being extracted was directly exchanged onto the resins packed in the expanded bed column. During this operation, the resins in chromatographic column were in an expanded status and there was space among resins. This character of expanded bed will allow particulate containing feed stock to go through the column without blockage. In the 1st dimension, the expanded bed was eluted by 2% aqueous NaCl solution and part of the fractions with shikimic acid was transferred to the interface unit-a sample loop for matching with the CCC solvent system.

In the 2nd dimension, the fractions (containing shikimic acid) were injected into the CCC column.The separation was performed to separate shikimic acid with a butanol-water-acetic acid (4:5:1) solvent system. 28-mg of shikimic acid was purified, with 99.6% purity and 50% recovery. This research case study proved the feasibility of the newly developed hyphenated chromatography method.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (No. 21006023) and the Fundamental Research Funds for the Central Universities. The authors would like to acknowledge an inspiring discussion with Prof. Ian Sutherland and Dr. Svetlana Ignatova at Brunel Institute for Bioengineering.

References

[1] Sutherland, I.; Fisher D. J. Chromatogr. A 2009, 1216 (4), 740-753. [2] Chase, H.A. Trends Biotechnol. 1994, 12 (8), 296–303.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-36 Comprehensive Separation of Lignans from Zanthoxylum planispinum by Counter-Current Chromatography Combined with High-Performance Liquid Chromatography/Tandem Mass Spectrometry Yanbin Lu*, Xianjiang Lin, Kuiwu Wang, College of Food and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China *fax +86-571-88071024-7587, e-mail: [email protected] Keywords: Counter-current chromatography; Comprehensive Separation; High-performance Liquid Chromatography/Tandem Mass Spectrometry; Zanthoxylum planispinum; Lignans.

An effective method combing fast counter-current chromatography (CCC) and HPLC/MS for comprehensive separation of antioxidative lignans from traditional Chinese medicine, Zanthoxylum planispinum Sied. Et Zucc., is presented.

The separation efficiency of CCC is largely depending on the selection of proper biphasic solvent system. One of the golden rules is that: the ratio of the two partition coefficients (K) or the separation factor (α = KD1/KD2, where KD1 >

KD2) ought to be greater than 1.5 in the semi-preparative multilayer separation column of a commercial HSCCC unit. Otherwise, the two components will be eluted together and formed only one peak in chromatogram. In the present work, in order to COMPREHENSIVE separation of as more as lignans from Z. Planispinum, several parametres, such as biphasic liquid systems, moblie phase flow rate, elution mode, etc., were carefully studied and optimzed. As a result, totally seven lignans were well separated in only one CCC process, and characterized by LC/MS/MS, exhibited great potential for natural drug discovery program of the present method.

Fig. 1 HPLC analysis of the crude extract.

Fig. 2 CCC separation of the target compounds with different conditions.

References

1. Y. Ito, J. Chromatogr. A 2005, 1065, 145-168. 2. Y. Lu, A. Berthod, Y. Pan, Anal. Chem. 2009, 81, 4048-4059. 3. Y. Lu, R. Hu, Y. Pan, Anal. Chem. 2010, 82, 3081-3085.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-37 Large-scale separation of alkaloids from Corydalis bungeana turca. by pH-Zone-Fefining counter-current chromatography Xiao Wang1, Donghongjing2,Yangbin1, Lin xiaojing2,Huangluqi1* 1. Institute of Chinese Medical Academy of Chinese Medical Science, 16 Dongzhimennei Street, Beijing 100700, China 2. Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China *Correspondence: Huang Luqi, Institute of Chinese Medical Academy of Chinese Medical Science, 16 Dongzhimennei Street, Beijing 100700, China. E-mail: [email protected] Fax: +86-10 -8402-7175 Keywords: pH-zone-refining counter-current chromatography; Large-scale separation; Corydalis bungeana; alkaloids Corydalis bungeana Turcz. (Papaveraceae) is a perennial herb distributed in many parts of the world, such as northern and eastern parts of China, the northern part of Korean peninsula, ect. (1). Its use for the treatment of influenza, upper respiratory tract infections, bronchitis, tonsillitis, acute nephritis and pyelonephritis have been reported in many studies (2), and pharmacological research showed that alkaloids are the main components (3, 4). Thus, it is necessary to establish an efficient method for the large-scale purification of alkaloids from Corydalis bungeana for further study of the biological activities of these alkaloids. pH-Zone-refining CCC was introduced by Ito as a novel preparative separation technique and has been used successfully for the large-scale purification of a varieties of natural products (5). The aim of this paper was to develop an efficient method for the large-scale separation of alkaloids from Corydalis bungeana Turcz. by pH-zone-refining CCC. The two-phase solvent system of pH-zone-refining CCC consisted of Pet–ethyl acetate–methanol–water 5:5:2:8 (v/v) where triethylamine (10 mM) was added to the upper organic stationary phase and hydrochloric acid (5 mM) to the aqueous mobile phase. 285 mg of compound A(protopine), 86 mg of compound B(corynoloxine), 430 mg of compound C(coryno1ine), and 115 mg of compound

Fig.1 pH-zone-refining counter-current chromatogram of crude alkaloids extracted from Corydalis bungeana D(acetylcoryno1ine) were obtained in one-step separation after an injection of 3.0 g of a crude extract, with purity of 99.1%, 98.3%, 99.0% and 98.5%, respectively, as determined by HPLC. The structures of the oxindole alkaloids were identified by ESI-MS, 1H NMR and 13C NMR. The overall results in our work clearly demonstrated that pH-zone-refining CCC is highly suitable for the requirements of large-scale isolation of alkaloids from natural plants. References

[1] Chen, X., Nigel, C. V., Peter, J. H., Monique, S. S., Phytochemistry 2004, 65, 3041–3047. [2] Qingdao TCM Research Group, 1972. Zhong Cao Yao Tong Xun 5, 16. [3] Xie, C. Kokubun, T., Houghton, P. J. Simmonds, M. D. J., phytotherapy research 2004, 18:497-500. [4] Yang J. G., Yuan H. N.,, Che J., Zhang Q., Journal of clinical pharmacology 1990, 6, 35–36. [5] Ito, Y., J. Chromatogr. A 2005, 1065, 145–168.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 O-38 Separation of the synthetic products of acetylated rhein by high-speed counter-current chromatography

Yindi Zhu, Ying Zhan, Haishui Gui, Qian Wei, Yangyang Liu, Tunhai Xu* Beijing University of Chinese Medicine * NO.11, East North 3rd Ring Road, Chao Yang District, Beijing, China; fax: 01064286935; E-mail: [email protected] Keywords: high-speed counter-current chromatography，1-acetylrhein，8-acetylrhein

Rhein is one of the main components in many traditional Chinese medicine as Rhubarb, Tuber Fleeceflower Root, Polygonum Cuspidate in polygonaceae plants, which has many pharmacological actions such as anti-tumor, anti-inflammatory, antifibrotic, anti-bacterial, immune suppression, diuretic and purgative, together with low toxicity and safety features, it has been concerned by many researchers [1]. Diacerein, the rhein acetylated synthetic product, which is a new type interleukin-21B (IL21B) inhibitor, can inhibit the activity of interleukin 21 (IL21) and IL2l synthesis in synovium [2]. For the similar structural among rhein acetylated synthetic products, especially the isomers of 1-acetylrhein and 8-acetylrhein, conventional chromatography is difficult to separate effectively, hinder the further activity study. In this study, high-speed counter-current chromatography (hsccc) was applied to seperate rhein, 1-acetylrhein, 8-acetylrhein and diacerein derivatives of rhein. Complete seperation of rhein, 1-acetylrhein, 8-acetylrhein and diacerein were achieved after one single separation by hsccc, and their purity of were more than 98% analyzed by HPLC, respectively.

Figure 1 Chromatogram of separation of the synthetic products of acetylated rhein by high-speed counter-current chromatography

References 1. YU Jia, WU Xiao-qing, SUN Hai-feng, et al. Advance of biological activity of rheinand itsderivatives. Pharmaceutical and Clinical Research, 2008, 16(2):125. 2. WANG Lin, MAO Yu-jia, WANG Wen-jie. Inhibitory effect of diacerein on osteoclastic bone destruction and its possible mechanism of action. Acta Pharmaceutica Sinica, 2006, 41 ( 6): 555- 560.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Authors Title Code

Preparative separation and purification of chemical Eli, Y.; Yang, Y.; Yili, A.; Aisa, H. compositions from Mentha longifolia L. by normal and P-1 A.* reverse phase high-speed counter-current chromatography Preparative separation and purification of five oxindole Fang, L.; Li, J.; Lin, Y.; Zhou, J.; alkaloids from Gelsemium elegans by pH-zone-refining P-2 Geng, Y.; Wang, X.* counter-current chromatography Faure, K.*; Mekaoui, N.; Berthod, Evaluation of Limonene as a possible “green” Non polar P-3 A. solvent for alkane replacement Gu, D.; Yang, Y.; Aisa, H. A., Ito, Novel design for centrifugal counter-current chromatography: P-4 Y. * Ⅵ. Ellipsoid column A new two-phase solvent system of chloroform/ acetyl acetate/ Han, Q. methanol/ water and its use in HSCCC separation of P-5 aconitines from aconite roots Isolation and purification of ecdysterone from Serratula Huang, J.; Xu, X.*; Xie, Z.; Yang, chinensis S. Moore by high-speed counter current P-6 M.; Xie, C. chromatography Dual-mode counter-current chromatography applied to Huang, X.; Chen, X.; Pei, D.; isolation maslinic and oleanolic acids from the olive milling P-7 Zhang, J.; Di, D. * subproduct Kim, T. B.; Kim, H. W.; Sung, S. Preparative separation of major constituents in OPuntia P-8 H.* ficus-indica by high-speed countercurrent chromatography Kitazume, E.*; Sudo, T.; Kubo, H.; Elimination of metal elements by high-speed counter-current P-9 Watanabe, M.; Ito, Y. chromatography by introducing air and solid absorbents Preparative isolation of chemical constituents from the roots of Lee, K. J.; Ha, I. J.; Kim, Y. S. * Phlomis umbrosa by high-speed counter-current P-10 chromatography Li, L.; Yang, Y.; Hou, X.; Ba, H.; Bioassay-guided separation and purification of water-soluble Gu, D. ; Abdulla, R.; Xin, X.; Wu, antioxidants from Carthamus tinctorius L. by combination of P-11 G.; Aisa, H.A. * chromatographic techniques Preparative isolation and purification of tanshinones from Liang, J.; Meng, J.; Wu, S.* Salvia miltiorrhiza Bunge by a novel counter-current P-12 chromatography with an upright conical coils Preparative separation of three flavonoids from the flower Liang, Y.; Ma, Y.; Ito, Y. * buds of Daphne genkwa by pH-zone-refining vortex P-13 counter-current chromatography

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Authors Title Code

Mekaoui, N.*; Faure, K.; Berthod, Multiple dual mode and trapping molecules for Coomassie P-14 A. brilliant blue G-250 purification Multi-dimension counter-current chromatography for high- Meng, J.; Wu, S.* P-15 throughput analysis of natural products Shiono, H.*; Matsui, T.; Okada, T.; Gentle and high-yield preparative separation method of P-16 Chen, H.M.; Ito, Y. basophils using a rotary-seal-free continuous-flow centrifuge Betalain separation from red beet juice (Reta vularis L.) by Spórna-Kucab, A.*; Ignatova, S.; high-performance countercurrent chromatography (HPCCC) P-17 Garrard, I.; Wybraniec, S. in polar high-salt solvent systems Separation of decarboxy-/dehydro- betalains by Spórna-Kucab, A.*; Ignatova, S.; high-performance countercurrent chromatography in ion-pair P-18 Garrard, I.; Wybraniec, S. solvent systems Wang, G.; Chen, X.; Huang, X.; Development of new pattern PTFE column of HSCCC P-19 Yun, L.; Di, D.* Gomathi,C.; Boustie, J.; Audo, Multple dual mode centrifugal partition chromatography as an G.* ; Ismail, N. H.; Quémener, C. efficient method for the purification of 4-phenylcoumarins P-20 L.; Awang, K. form mesua elegans Preparative separation of two subsidiary colors of FD & C Weisz, A.*; Roque, J. A.; Mazzola, yellow No. 5 (Tartrazine) by spiral high-speed counter-current P-21 E. P.; Ito, Y. chromatography Novel sample pretreatment method for increasing the Yang, Y.; Gu, D.; Chen, Q.; Yili, preparative scale of pH-zone refining counter-current P-22 A.; Aisa, H. A.* chromatography Solvent gradient counter-current chromatography purification Yang, Z.; Wu, S.* P-23 of podOPhyllotoxins from Dysosma versipellis (Hance) Yoo, G.; Kim, T. B.; Yang, H.; Preparative separation of isoquinoline alkaloids from COPtis P-24 Park, J. H.; Kim, Y. C.; Sung, S. H. japonica by high-speed counter-current chromatography Zhang, J.; Huang, X.; Pei, D.; Screening for anti-diabites fraction in the leaves of Olea P-25 Chen, X.; Di, D.* europaea L. by HPCCC Han,T.; Heuvel, R.; Sutherland, I.; Flow separation of cells in counter-current chromatography P-26 Fisher, D. (CCC) centrifuges Erastov, A. A.; Yulya, A.; Comparative study of three modifications of the Zakhodjaeva, Y. A.; Voshkin, A. A. P-27 controlled-cycle liquid-liquid chromatography device Kostanyan, A. E.*

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Authors Title Code

Qiu, F.; Friesen, J. B., McAlpine, J. Targeted analysis of natural products by K-based P-28 B.; Lankin, D. C.; Pauli, G. F.* countercurrent separation van den Heuvel, R. N. A. M.; Characterisation of quaternary Heptane-Water based Harris, G.; Douillet, N.; Thickitt, phasesystems across the polarity range P-29 C.; van den Heuvel, E. A.; forCounter-currentChromatography Ignatova, S. Vieira, M. N.; Welke, K.; Large-Scale Preparative Profiling of Anacardic Acids in Murillo-Velásquez, J. A.; Cashew Nut Shell Oil Liquid by Spiral-Coil Countercurrent P-30 Leitão, S. G.; Winterhalter, P.*; Chromatography and Off-Line ESI-MS-MS Continuous Jerz, G. Infusion Bonsch, B.; Dillschneider, R.; Preparative Metabolite Profiling of the Diatom Microalga Rosello, R.; Posten, C.; Phaeodactylum tricornutum by High-Speed Countercurrent P-31 Winterhalter, P.*; Jerz, G. Chromatography and Continuous APCI-MS-MS Infusion Zebrafish bioassay-guided isolation of anti-angiogenic Han, L. W.; Yuan, Y. Q.; He, Q. X.; components by high-speed counter-current chromatography P-32 Chen, X.Q.; Liu, K.C.* from licorice High-speed counter-current chromatography preparative Li,Y. l.*;Liu, Y. l.; Wang, P.; Chen, isolation and purification of four xanthone clycosides from T.; Zhao, X.; Chen, C.; Sun ,J. P-33 tibetan medicinal plant Halenia elliptica

A Combination of Ultrahigh pressure-Assisted Extraction with Lin, X. J.; Dong, H. J.; Geng, Y. L.; High-Speed Counter-Current Chromatography for the P-34 Liu, F.; Wang, D. J.; Wang, X.* Preparation of Cantharidin from the blister beetle Continuous separation of capsaicin and dihydrocapsaicin using Goll, J.; Frey, A.; Minceva. M.* P-35 sequential centrifugal partition chromatography Li, Y.*; Buonocore, F.; Barker, J.; Separation of six ginsenosides from panax ginseng using Barton, S. J.; Sutherland, I.; P-36 high-performance counter-current chromatography Ignatova, S. Jiang, W.; Wu, B.* Isolation and Purification of Pheromones from Ground Beetles P-37 by Counter-Current Chromatography

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Authors Title Code

Ye, Q. X.; Yin, S.; Zhao, Z. M.; Separation of bioactive compounds from Dark tea using Yang, D. P.; Zhu, L. P.; Brown, P-38 HSCCC target-guided by α-glucosidase inhibitory activity L.; Wang, D. M.* Application of high speed countercurrent chromatography in Gök, R.; Jerz, G.; Winterhalter, P.* P-39 organic synthesis purification Okunji, C. O.; Awachie, P. I.; Preparative separation of biflavonoids from Garcinia Kola Iwu, M. M.; Tchimene, M.; Ito, heckel .seeds by spiral high-speed counter-current P-40 Y.* chromatography Separation of anti-tumor constituents from KalOPanax Xu, J.; Cao, X. L.*; Yin, L.; Cheng, septemlobus (Thunb.) Koidz by silica gel column and P-41 C.; Ren, H. high-speed counter-current chromatography Effective and Preparative Separation of Bioactive Flavonoids Chen, Z. ; Lu, Y. B., * from Gynostemma pentaphyllum Tea Using Elution-Extrusion P-42 Counter-Current Chromatography Preparative separation of polyphenols by high-speed Li, Z. H.; Li, M. H.* P-43 counter-current chromatography Brownstein, K.; Rottinghaus, G. Isolation of the cycloartane glycosides from Sutherlandia P-44 E.; Knight, M.; Ito, Y.; Folk, W. R.* Frutescens by HSCCC using the spiral tubing support column Liu, D. H.; Shu, X. K.; Liu, W.; Preparative separation of alkaloids from Aconitum carmichaeli P-45 Wang, X.*; Yang, B.; Huang, L. Q. by pH-zone-refining counter-current chromatography Preparative separation and purification of hypocrellins from Xu, M. X.; Liu, F.; Ma, X. L.; Shiraia Bambusicola by high-speed counter-current P-46 Zhao, H. Q.; Liu, J. H.; Wang, X.* chromatography and preparative high performance liquid chromatoraphy Separation ,identification and purfication of active constituents Gao, F. Y.; Fang, G.; Zhou, T. T.; of Polygonum cuspidatum Sieb. et Zucc. by P-47 Li, J.; Fan, G. R.* HPLC-PDA-ESI-MSn and HSCCC Grudzien, L.; Fisher, D.; Madeira, Purification of a small lectin from a L.; Ma, J.; Sutherland, I.; Garrard, hydroponic culture media using P-48 I. centrifugal partition chromatography

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 Authors Title Code

Duan, P. X.; Xu, Q. Q.; Zhang, L.; Purification of luteim from kale by high speed countercurrent Wang, X. D. chromatography P-49

New hyphenated CPC-HPLC-DAD-MS strategy for Michel, T.; Destandau, E.; Fougère, Simultaneous Isolation, Analysis and Identification of P-50 L.; Audo, G.; Elfakir, C. Phytochemicals. Impurities preparaton and identification of sodium tanshinone Chen, S. Q.; Zhou, T. T. Liu, C. C.; IIA sulfonate by HSCCC and LC-MSn P-51 Fan, G. R.

Method optimisation for isolating purified α-mangostin from Murhandini, S.; Keaveney, E.; garcinia mangostana l. Rinds using HPCCC Barton, S. J.;Barker, J.; Garrard,I.2; P-52 Fisher, D.; Ignatova, S.

Li, J. L.; Fang, L.; Wang, D. J.; Preparative isolation and purification of biflavonoids from Shu, X. K.; Geng, Y. L.; Wang, X.* ephedra sinica by high-speed counter-current chromatography P-53

Wang, C.; Chao, Z.; Sun, W.; Isolation of Chemical Constituents from Ilex rotunda by Wu, X.; Ito, Y. b High-Speed Counter-Current Chromatography P-54

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-1 Preparative separation and purification of chemical compositions from Mentha longifolia L. by normal and reverse phase high-speed counter-current chromatography Yusupjan Eli a,b, Yi Yang a, Abulimiti Yili a, Haji Akber Aisa a,* a Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China *Corresponding author: Haji Akber Aisa, Tel: +86 991 3835679, Fax: +86 991 3838957, E-mail: [email protected]

Keywords: crude sample preparation, yield, pH-zone refining counter-current chromatography High-speed counter-current chromatography (HSCCC) has been widely applied for the separation and purification of biological samples with two-phase solvent systems [1-3]. However, the natural products or their fractions are constituted by complex mixtures. Usually, the studies focus on separation of compounds in the crude fraction with bioactivity from herb plant. In this fraction, some constituents are hydrophobic and some are hydrophilic. When the normal phase HSCCC was used for separation of hydrophobic compounds with appropriate K values, the retention time of hydrophilic compounds were very big because of the large K values, and vice versa. So normal and reverse phase HSCCC can be combined for the comprehensive investigation of chemical compositions in the active fraction. Mentha longifolia L. is a traditional Uighur medicine for the treatment of gastritis, carminative, headache, curette, sinusitis, hemorrhoids, diuretic, menstruate etc [4]. However, scientific data concerning the basic composition are lacking. It encouraged us to perform the phytochemical investigation for Mentha longifolia L. In the study, five compounds were isolated from Mentha longifolia L. by normal and reverse phase HSCCC. The normal and reverse phase HSCCC separations were performed with two biphasic solvent systems composed of chloroform-methanol-water (4:3:2, v/v) and n-hexane-ethyl acetate-methanol-water-acetic acid (1:10:1:10:0.25, v/v), respectively. As a result, 11.8mg of diosmetin, 5.3mg of acacetin and 4.6mg of apigenin were obtained from 200 mg ethyl acetate extracts of Mentha longifolia L. in normal phase elution mode by HSCCC. And 6.1 mg of rosmarinic acid and 3.2 mg caffeic acid were obtained from 200 mg ethyl acetate extracts of Mentha longifolia L in reverse phase elution mode by Figure 1. The structures of isolated compounds from Mentha longifolia L. by normal and reverse HSCCC. Their structures （Fig. 1）were identified by MS phase high-speed counter-current and NMR. chromatography. References To be ordered numerically following the appearance in the text with this format: Arial normal letter font 8. No space between references. Citation style follows JACS/ACS format, an Endnote style file can be obtained from www.ccc2006.org. 1. Ito, Y. J. Chromatogr. A 2005, 1065, 145-168. 2. Berthod A; Maryutina T; Spivakov B; Shpigun O; Sutherland I A. Pure Appl. Chem. 2009, 81, 355-387. 3. Yang Y; Huang Y; Gu D; Yili A; Sabir G; Aisa H A. Chromatographia 2009, 69, 963-967. 4. Sabir H; Kadir A. Encyclopedia of Uighur medicine, science technology publishing company of Shanghai, 2005, 177.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-2 Preparative separation and purification of five oxindole alkaloids from gelsemium elegans by ph-zone-refining counter-currentchromatography Lei Fang, JiaLian Li, YunLiang Lin, Jie Zhou, YangLing Geng, Xiao Wang* Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China *Correspondence: Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China. E-mail: [email protected] Fax: +86-531-8296-4889

Keywords: Gelsemium elegans; Oxindole alkaloids; pH-zone-refining counter-current chromatography; Gelsemium elegans Benth. (Loganiaceae) is known as a toxic herb grown in the south of China and has been used traditionally for the treatment of pain, spasticity, and skin ulcers in Chinese folkloric medicine (1). The pharmacologically active constituents of G. elegans mainly consist of oxindole alkaloids, and more than 70 alkaloids based on six different structural skeletons have been isolated from G. elegans (2,3).These alkaloids demonstrate potent cytotoxic, immunomodulating and antiarrhythmic, analgesic, and anti-inflammatory activities (4,5). Considering such various biologically activities of oxindole alkaoloids, it is necessary to develop an efficient method to separate and purify large quantities of single oxindole alkaoloid with high purity for further pharmacological research.

In this research, pH-zone-refining counter-current chromatography was successfully applied to the preparative separation and purification of five oxindole alkaloids from the stems of G. elegans. The experiment was performed with a two-phase solvent system composed of petroleum upper organic stationary phase as a retainer and 10mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter. 3.0 g of the total alkaloid was purified in one-step separation, yielding 110 mg of 19-xo-gelsenicine, 126 mg of gelsemine, 302 mg of gelsevirine, 107 mg of gelsenicine and 89 mg of humantenine with the purities of 98.1%, 98.5%, 98.7%, 98.4% and 98.8%, respectively, as Figure 1. Separation of six oxindole alkaloids from determined by high-performance liquid chromatography. The structures of the oxindole the stems of G. elegans by pH-zone-refining CCC. alkaloids were identified by UV, ESI-MS, 1H NMR Solvent system: petroleum ether–ethyl and 13C NMR. acetate–methanol–water; sample size: 3.0 g; flow-rate: 1.5 ml/min; detection: 254 nm; revolution speed: 800 rpm. a: 19-xo-gelsenicine; b: gelsemine; c: gelsevirine; d: gelsenicine; e: humantenine ether–ethyl acetate–methanol–water (3:7:1:9, v/v), where 10mM triethylamine (TEA) was added to the This is the first report that oxindole alkaloids from the stems of G. elegans were successfully isolated and purified by the pH-zone-refining counter-current chromatography method, which demonstrated that pH-zone-refining CCC is a fast, effective and powerful technique for the isolation and purification of alkaloids from natural herbs. References

1. Editorial committee of Chinese Materia Medica. Chinese Materia Medica, Vol.17; Shanghai Science and Technology Press: Shanghai, 1999; p. 213 2. Xu, Y. K.; Yang, S. P.; Liao, S. G.; Zhang, H.; Ling, L. P.; Ding, J. J.; Yue, M. J. Nat. Prod. 2006, 69, 1347–1350. 3. Kogure, N.; Ishii, N.; Kitajima, M.; Wongseripipatana, S.; Takayama, H. Org. Lett. 2006, 8, 3085–3088. 4. Rujjanawate, C.; Kanjanapothi, D., Panthong, A. J. Ethnopharmacol. 2003, 89, 91–95. 5. Kitajima, M.; Nakamura, T.; Kogure, N.; Ogawa, M.; Mitsuno, Y.; Ono, K.; Yano, S.; Aimi, N.; Takayama, H. J. Nat. Prod. 2006, 69, 715–718.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-3 Evaluation of Limonene as a possible "green" non polar solvent for alkane replacement Karine Faure, Nazim Mekaoui, Alain Berthod Institut des Sciences Analytiques, University of Lyon, Villeurbanne, France *fax +33 472 448 319, e-mail: [email protected] Keywords: green solvent, alkane substitution, limonene

Alkanes are highly used as solvent in CCC for their low polarity and total insolubility with water. Produced by petroleum industry, alkanes are non renewable and pollutant. To develop ecofriendly technologies, the policy is to favor bio-renewable solvents. Limonene is, with the possible exception of α-pinene, the most frequently occurring natural monoterpene. It is a major constituent (90-95%) of the orange essential oil. Since it comes from food waste, its use in industry is highly plebiscited. Following Friesen [1] that first proposed Limonene as a possible renewable solvent in CCC liquid system formulations, limonene was tested for the replacementof heptane in the simple solvent system heptane/methanol/water.

Figure 2. Separation of methylparaben – diethyl phthalate on a 38 Figure 1. Limonene represents ml hydrostatic instrument (Kromaton R2D2). Limonene-MeOH/water 90/10 ascending mode with upper aqueous 90-95% of orange essential oil mobile phase, 2ml/min, 2500 rpm. Sf = 52%, P = 15 bars.

The Friesen initial results were confirmed: in hydrodynamic instruments, the very low density difference between limonene (lower phase) and the methanol/ water mixture resulted in a constant flooding and poor or nearly no phase retention (small Sf). The use of limonene and heptane blends could help but it conflicts with the environmental concern. The new finding is that hydrostatic instruments (centrifugal partition chromatographs) could easily retained limonene as stationary phase, with Sf reaching values as high as 60%. The low density difference is actually an advantage leading to reduced working pressures (around 15 bars), allowing for the user to work at high rotor rotation speeds and tight liquid stationary phase holding. When compared with heptane in hydrostatic instrument, limonene was less retained as stationary phase (60% instead of 70%) but the partition coefficients of test solutes were slightly higher when using limonene. The overall resolution was better with limonene than with heptane. Since the addition of water increases the density difference in this solvent system, it was even possible to modulate the retention of solutes by changing the water content in the mobile phase. This work is a first investigation with high hopes to develop new greener solvent systems for CCC. Reference 1. J.B. Friesen, Use of renewable solvents in the formulation of CCC separation systems, Communication 4-5, CCC 2010, The 6th International Symposium, Lyon, France, 2010,

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-4 Novel design for centrifugal counter-current chromatography: Ⅵ. Ellipsoid column Dongyu Gu a, b, Yi Yang a, b, Haji Akber Aisa b, Yoichiro Ito a,* a Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA b Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China *Corresponding author: Yoichiro Ito, Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, Bldg.10, Room 8N230, Bethesda, MD 20892-1762, USA Keywords: ellipsoid column, hydrostatic counter-current chromatographic system, centrifugal counter-current chromatography, retention of the stationary phase, peak resolution

In our previous studies, a series of novel column designs has been introduced to further improve the performance of Hydrostatic counter-current chromatography (CCC) which can produce highly efficient analytical separations [1], including triangular coil [2], zigzag [3], saw tooth [4] and figure-8 columns [5]. Recently, we found that the highest resolution (Rs) was obtained when the figure-8 column was mounted parallel to the radially acting centrifugal force field. The performance of the system was examined in various test samples [6,7]. The high partition efficiency of this system may be explained by hydrodynamic motion and interaction of the two phases in the column at various angles. When the column angle is set at 0º against the radially acting centrifugal force field, the second loop of figure-8 provides two separate partition segments for droplet flow to improve Rs (Fig. 1A) [7]. Based on this finding, the ellipsoid column is designed in the present study, which can provide a much longer partition segment than that in the figure-8 column (Fig. 1B). The performance of the ellipsoid column with a capacity of 3.4 mL was examined with three different solvent systems composed of 1-butanol-acetic acid-water (4:1:5, v/v) (BAW), hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMH), and 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in water (PEG-DPP) each with suitable test samples. In dipeptide separation with BAW system, both stationary phase retention (Sf) and Rs of the ellipsoid column were much higher at 0º column angle than at 90º column angle, where elution with the lower phase at a low flow rate produced the best separation yielding Rs at 2.02 with 27.8% Sf at a flow rate of 0.07 ml/min. In the DNP-amino acid separation with HEMW system, the best results were obtained at a flow rate of 0.05 ml/min with 31.6% Sf yielding high Rs values at 2.16 between DNP-DL-glu and DNP-β-ala peaks and 1.81 between DNP-β-ala and DNP-L-ala peaks. In protein separation with PEG-DDP system, lysozyme and myolobin were resolved at Rs of 1.08 at a flow rate of 0.03 ml/min with 38.9% Sf. Most of those Rs values exceed those obtained from the figure-8 column under similar experimental conditions previously reported. References 1. Y. Ito, R.L. Bowman, Science 167 (1970) 281. 2. Y. Ito, H. Yu, J. Liq. Chromatogr. Rel. Technol. 32 (2009) 560. 3. Y. Yang, H. A. Aisa, Y. Ito, J. Liq. Chromatogr. Rel. Technol. 32 (2009) 2030. 4. Y. Yang, H. A. Aisa, Y. Ito, J. Liq. Chromatogr. Rel. Technol. 33 (2010) 846. 5. D. Gu, Y. Yang, J. D. Eng, H. A. Aisa, Y. Ito, J. Liq. Chromatogr. Rel. Technol. 33 (2010) 572. 6. Y. Yang, D. Gu, H. A. Aisa, Y. Ito, J Chromatogr. A 1218(2011)6128. 7. Y. Yang, D. Gu, H. A. Aisa, Y. Ito, J Chromatogr. B 879(2011)3802.

Figure 1. Hydrodynamic motion and interaction of the two phases in the figure-8 and ellipsoid column. The column was parallel to the acting centrifugal force

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-5 A new two-phase solvent system of chloroform/acetyl acetate/methanol/water and its use in HSCCC separation of aconitines from aconite roots Quan-Bin Han* School of Chinese Medicine, Hong Kong Baptist University *[email protected] Keywords: two-phase solvent system， HSCCC， aconitines

High-speed counter-current chromatography (HSCCC) has been popularly used to prepare high-purity natural compounds in laboratories [1,2]. The unique mechanism makes it a magic and powerful separation technique in which two-phase solvent system plays a crucial role. Basically, HSCCC is a continued liquid-to-liquid solvent partition where the target compounds are competitively distributed between the two-phase solvents due to their different partition coefficients (K values). The competition reaches a climax when K value is 1 which means the two-phase solvents exhibit equal ability to attract the target compound.

Figure 1. HSCCC Chromatogram of CaoWu extract. Solvent system: Chloroform-Ethyl acetate-MeOH-0.3M HCl in stepwise elution; stationary phase: upper phase of 1.5:1:1.5:2; mobile phase: lower phase of 1.5:1:1.5:2 in 0-250 min,lower phase of 2.75:1:1.5:2 in 250-400 min, lower phase of 3:0.5:1.5:2 after 400 min;revolution speed: 800 rpm; separation temperature: 25ºC; sample size: 102.24 mg; detection wavelength: 230nm.

Many different two-phase solvent systems have been developed in order to separate the highly diverse natural compounds [3]. Aqueous two-phase solvent systems like were des igned for macromolecules [4-9]. Regarding the separation of small molecules, two-phase solvent systems composed of organic solvents and water are commonly used, for example, the most popular system hexane/acetyl acetate/methanol/water [3]. The lower phase is always aqueous. Of course, there is an exception, namely chloroform/methanol/water in which the lower phase is organic solvent because chloroform contains higher density.

Selection of a proper two-phase solvent system is definitely the most important step in a successful HSCCC separation. At the same time, it is also time consuming. It is hard to make water and oil contain similar dissolving capacities. We reports in this paper a new two-phase solvent system in which both phases can contain sufficient organic solvents to balance their dissolving capacities. Its two-phase forms based on not only the water-oil difference but also the difference of density. It is chloroform/acetyl acetate/methanol/water. Different from chloroform/methanol/water, the upper phase contains more organic solvent acetyl acetate. Its polarity is close to chloroform, but its density is much smaller. It is easier to adjust the new solvent system to get satisfactory K values. And it succeeded in preparation of conitine, hypaconitine, mesaconitine from the crude aconite roots (Figure 1), benzoylmesaconine from the processed aconite roots, and yunaconitine from A. forrestii, where the commonly used hexane/acetyl acetate/methanol/water failed.

References 1. Y. Ito, J. Chromatogr. A. 1065 (2005) 145. 2. A. Marston, K. Hostettmann, J. Chromatogr. A. 1112 (2006) 181. 3. X.L. Cao, High-speed counter-current chromatography and 4. its application, Chemical Industry Press, Beijing, 2005, 390. 5. C.W. Shen, T. Yu, J. Chromatogr. A. 1151 (2007) 164. 6. Y. Shibusawa, N. Takeuchi, K. Sugawara, A. Yanagida, H. Shindo, Y. Ito, J. Chromatogr. B. 844 (2006) 217. 7. Z.M. Chao, Y. Shibusawa, H. Shindo, Y. Ito, J. Liq. Chromatogr. Rel. Technol. 26 (2003) 1895. 8. X. J. Sun, B. Tang, Y. M Xu, Chin. Patent 2008 CN 101323648. 9. G. L. Song, Q. Z. Du, J. Chromatogr. A. 1217 (2010) 5930. 10. Z.G. Jiang, Q.Z. Du, L.Y. Sheng, Fenxi Huaxue 37 (2009), 412. 7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-6 Isolation and purification of ecdysterone from Serratula chinensis S. Moore by high-speed counter current chromatography Jieyun Huang1, Xinjun Xu*, Zhisheng Xie1, Mei Yang1, Chunyan Xie1 School of Pharmaceutical Sciences, Sun Yat-Sen University * No. 132, East Waihuan Rd.,Guangzhou Higher Education Mega Center, 510006 Guangzhou, China e-mail address: [email protected]. fax: +86 020 39943041. Keywords: Serratula chinensis S. Moore, HSCCC, ecdysterone

Serratula chinensis S. Moore (Guang Sheng Ma) is a perennial herbaceous plant growing mainly in South China [1]. Ecdysterone is the major component of Serratula chinensis S. Moore [2]. In this paper, ecdysterone was isolated and purified from Serratula chinensis S. Moore by high-speed counter-current chromatography with a solvent system of ethyl acetate-n-butanol-water (4:1:5, v/v/v) in one step. From 200 mg of the n-butanol extract of Serratula chinensis S. Moore, 23 mg of ecdysterone was obtained with purity of 99.3%, as determined by HPLC. The structure was identified by MS, UV, and NMR analysis. In this study, a rapid method for isolation and purification of ecdysterone from Serratula chinensis S. Moore extract was established.

The separation by HSCCC depends largely on a suitable partition coefficient (0.5≤K≤2.0) [3]. According to the the polarity of ecdysterone, n-butanol-water was used to test the K value firstly. But the K value measured by HPLC of n-butanol-water was too high, indicating that it was not a suitable system for the separation. So ethyl acetate was added to adjust the K value. Then the K value of ethyl acetate-n-butanol-water was tested and proved to be better. In order to optimize separation procedure and get pure compound, ethyl acetate-n-butanol-water with different volume ratios were tested. No.2 and No.3 was not suitable because the K values were still high. Experiments showed that No.4 could efficiently separate ecdysterone. The K value of No.4 was 0.90 and it was very suitable to separate the target compound 23 mg of ecdysterone was obtained from 200 mg of Serratula chinensis S. Moore n-butanol extract. Therefore, No.4 was chose as the solvent system for HSCCC.

Table 1. The K value of the targeted component measured in different solvent systems

Ratio(v/v/ No. Solvent system K v) No.1 n-butanol-water 1:1 5.03 Ethyl No.2 acetate-n-butanol- 2:3:5 3.93 water Ethyl Figure1. HSCCC chromatogram of the n-butanol extract of No.3 acetate-n-butanol- 3:2:5 2.48 Serratula chinensis S. Moore. The column volume was 120 mL. water Solvent system: ethyl acetate-n-butanol -water (4:1:5, v/v/v). The upper phase served as the stationary phase. The lower phase (mobile Ethyl phase) was pumped into the column from the head-to-tail at a flow No.4 acetate-n-butanol- 4:1:5 0.90 rate of 2 mL·min-1. The rotation speed was 860 rpm. Retention of water the stationary phase: 54.2%. Injection volume: 10 mL; the wavelength: 254 nm; fraction collector: 3 min for each tube. References

1. Ling, T. J.; Xia, T.; Wan, X. C.; Li, D. X.; Wei, X. Y.. Cerebrosides from the roots of Serratula chinensis. MOLECULES. 2006, 11, (9), 677-683. 2. Cai, Q.; Zeng, J.; Lin, S., Studies on Quality Standard of Radix Serratulae Chinensis. Journal of Fujian University of TCM. 2011, 21, (4), 38-41. 3. Li, L.; Tsao, R.; Yang, R.; Liu, C.; Young, J. C.; Zhu, H. Isolation and purification of phenylethanoid glycosides from Cistanche deserticola by high-speed counter-current chromatography. Food Chemistry. 2008, 108, (2), 702-710.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-7 Dual-mode counter-current chromatography applied to isolation maslinic and oleanolic acids from the olive milling subproduct Huang Xin-Yi1, Chen Xiao-Fen1,2, Pei Dong1, Zhang Jia1, Di Duo-Long1* 1 Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000 2 Graduate University of the Chinese Academy of Sciences, Beijing 100049 *Corresponding author fax: 0931-4968094; e-mail: [email protected] Keywords: Dual-mode counter-current chromatography, maslinic acid, oleanolin acid

Maslinic acid (MA) and oleanolic acid (OA) are pentacyclic triterpene derivatives which have recently caused great interest due to their many biological effects, such as antioxidant activity, anti-HIV activity, anti-inflammatory and supressive effects on glycogen phosphorylase, cancer cell, hyperlipidemia, and so on. Olive fruit has high contents of MA and OA. In tis work, MA and OA have been isolated and puried by dual-mode counter current chromatography (CCC) from olive milling subproduct. It is first and important step to select a suitable two-phase solvent system in a CCC separation. A classical solvent system consist of n-hexan-ethyl acetate–methanol –water (HEMW) was used to achieve this separation. A series of HEMW biphasic systems with different composition were screened to optimize the solvent system for CCC separation and their partition coefficients (k) values of the target compounds were measured and summarized in Table 1. Given the enormous range of k values of MA and OA, the dual-mode (DM) was used to elute the two compoents. The separation was achieved on a DE Spectrum HPCCC under analysis condition. The upper phase of biphasic system was as stationary phase and moble phase was bumped at a flow rate of 1.0 mL/min with rotation at 1500 rpm and at RP mode. When the MA was eluted, the elution modes were changed from initial RP to NP by switching the positionof the valves. The effluent was collected each minute and analysed by HPLC. The both purities of MA and OA were more than 90%.

Table 1. The k values of MA and OA in different solvent systems. Solvent system k value composition (v:v) MA OA 6:1:6:1 0.077 0.317 6:2:6:1 0.135 0.320 6:2:4:1 0.182 0.702 6:2:4:2 0.385 2.882 6:2:3:1 0.223 1.071 6:2:2:1 0.993 9.369

Acknowledgements: The authors gratefully acknowledge financial support by the ‘Hundred Talents Program’ of the Chinese Academy of Sciences (CAS), the National Natural Sciences Foundation of China (NSFC No. 20974116, 21175142).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-8 Preparative separation of major Constituents in Opuntia ficus-indica by high-speed countercurrent chromatography Tae Bum KIM, Hyeon Woo KIM, Sang Hyun Sung* College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea *Corresponding author : [email protected] Opuntia ficus-indica (Cactaceae), found on Jeju Island (Republic of Korea), was reported for a rich source of flavonoids (1, 2). Narcissin (isorhamnetin-3-rutinoside) and other flavonoids were separated by High-speed countercurrent chromatography. The two-phase solvent systems comprising methylene chloride-methanol-n-butanol-water (5:4:3:5, v/v/v/v) and n-hexane-n-butanol-water (3:3:5, v/v/v) were employed to HSCCC. Isolated compounds were elucidated by ESI-MS and NMR techniques. The purity of compounds was evaluated to be over 90 % by HPLC-DAD.

Table 1. The K value of narcissin in different solvent system Solvent system K value

CHCl3-MeOH-H2O(4:1:2, v/v/v) 19.16

CHCl3-MeOH-H2O (3:4:2, v/v/v) 4.25

CH2Cl2-MeOH-BuOH-H2O (4:4:3, v/v/v) N/A

CH2Cl2-MeOH-BuOH-H2O (3:1:1:3, v/v/v/v) 2.26

CH2Cl2-MeOH-BuOH-H2O (3:1:2:3, v/v/v/v) 2.10

CH2Cl2-MeOH-BuOH-H2O (3:2:1:3, v/v/v/v) 2.90 Figure1. Separation diagram elucidated by ESI-MS and NMR techniques. CH2Cl2-MeOH-BuOH-H2O (5:4:3:5, v/v/v/v) 1.33 The purity of compounds was evaluated to be over 90 % by HPLC-DAD.

Figure 2. Major flavonoids in Opuntia ficus-indica

Figure 3. HSCCC chromatogram of Opuntia ficus-indica

References 1. Lee, E. H.; Kim, H. J.; Song, Y. S.; Jin, C.; Lee, K.; Cho, J.; Lee, Y. S. Arch. Pharm. Res. 2003, 26.

2. Jeong, S.J.; Jun, K. Y.; Kang, T. H.; Ko, E. B.; Kim, Y. C. Saengyak Hakhoechi. 1999, 30. Figure 4. HPLC-DAD chromatogram of flavonoids in Opuntia ficus-indica

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-9 Elimination of metal elements by high-speed counter-current chromatography by introducing air and solid absorbents

Eiichi Kitazume1,*, Tomoaki Sudo1, Hanae Kubo1, Miki Watanabe1 Yoichiro Ito2

1Faculty of Humanities and Social Sciences, Iwate University, Morioka, Iwate, 020-8550 Japan, Fax: +81-196-21-6825, e-mail:[email protected] 2Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892

Keywords: counter-current chromatograph, enrichment, metal elements

Radioactive materials were widely spread in the atmosphere from the nuclear power plant in Fukushima, Japan, following the strong earthquake on March 11, 2011. Now, cesium 137 with a half-life of 30 years especially poses a problem. Since a large area of soil and water has been polluted near the nuclear power plant, immediate measures for decontamination are urgently needed. Although there are various decontamination methods, for example by washing buildings and their roofs with high-pressure water-drainage equipment, the contamination is only washed away and allowed to spread, making this an invalid method to solve this problem. Moreover, using the ground decontamination method in which the top-soil is removed, the problem is now how to store or dispose the removed top-soil. Ideally the best method would be to condense the released radioactive materials into one spot and (if possible) get Ali Baba’s Genie to return things to their original state. Yet, this is far from realistic. Although high-speed counter-current chromatography (HSCCC) has so far been used to continuously condense impurities in a liquid phase without dispersing them, the method may also be used for efficient decontamination of metal elements including radioactive materials. Then, it may be possible to operate HSCCC with air, liquids and particles in the column in order to condense the target substance at the air-liquid interface or onto the solid phase. In our present study, the efficiency of removing substances by HSCCC is examined using air or air and adsorbent (CDP silica or cyclodextrin wrapped in a silica skeleton) in a column. The effectiveness of this novel HSCCC method is investigated by continuously introducing water containing metal elements (which simulates contaminated water) through the column.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-10 Preparative isolation of chemical constituents from the roots of Phlomis umbrosa by high-speed counter-current chromatography

Kyoung Jin Lee, In Jin Ha and Yeong Shik Kim

Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Korea **Fax: +82 2 765 4768599. E-mail: [email protected] (Prof. Yeong Shik Kim)

Keywords: Phlomis umbrosa ; phenylethanoid glycosides ; high-speed counter-current chromatography ;

HPLC-ELSD, HPLC-MS/MS

Phlomis umbrosa Turcz is a kind of perennial herbaceous plant growing in Korean peninsula and Northern part of China. In Korea its roots have been used traditionally for nourishing the kidney and consolidating essence, thus activating brain function, promoting memory and lengthening life span. This study focus on developing an effective method for isolating secondary metabolites from the roots of Phlomis umbrosa by high-speed counter-current chromatography. The 70% ethanol extract was loaded onto an open column packed with Diaion

HP-20 (resin) and fractionated by a methanol and water gradient elution. 5 compounds (one iridoid glycosides and four phenylethanoid glycosides) were separated from the 70% methanol fraction by HSCCC-ELSD with a two phase solvent system composed of ethyl acetate-buthanol-water (1:4:5, v/v). For reducing the separation time the flow rate of mobile phase was gradiently increased at specific time intervals. The purity of the isolated compounds was determined by HPLC-UV and ELSD.

The compounds purified in this study were; [1] acetyl shanziside methyl ester [2] apiosylverbascoside [3] alyssonoside [4] verbascoside [5] betonyoside D. Molecular structrues of these compounds have been identifided by electrospary ioinzation mass spectrometry (ESI-MS), 1H NMR and 13C NMR.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-11 Bioassay-guided separation and purification of water-soluble antioxidants from Carthamus Tinctorius L. by combination of chromatographic techniques Lingnan Li a, b, Yi Yang a, Xueling Hou a, Hang Ba a, Dongyu Gu a, Rahima Abdulla a, Xuelei Xin a, Guirong Wu b, Haji Akber Aisa a, * a Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China b School of Pharmacy, Xinjiang Medical University, Urumqi 830054, China *Corresponding author: Haji Akber Aisa, Tel: +86 991 3835679, Fax: +86 991 3838957, E-mail: [email protected] Keywords: Bioassay-guided separation, combination of chromatographic techniques, high-speed counter-current chromatography, Carthamus tinctorius L., water-soluble antioxidants. The flowers of Carthamus tinctorius L. (safflower), have been used as Chinese traditional medicine for over 2500 years and for the treatment of stroke, coronary heart disease, and angina pectoris [1,2]. In our study, the extract of safflower showed the antioxidant activity prompted us to investigate the chemical constituents and its association with antioxidant. But most of active compounds with high hydrophilicity in the active fraction are minor ingredients. The simultaneous preparation of major compounds and separation of these minor active compositions is a very big challenge, especially for water-soluble minor compositions. In order to investigate the minor water-soluble antioxidants, in the present study, a combined chromatographic method using high-speed counter-current chromatography (HSCCC) which has been widely used for various natural compounds separation [3,4] and Sephadex LH-20 chromatography was established for bioassay-guided separation and purification of water-soluble antioxidants from of Carthamus tinctorius L. florets. Following an initial cleanup step on the AB-8 macroporous resin, the crude sample Ⅱ was obtained and exhibited a potential ABTS radical cation scavenging activity with the SC50 value of 49.28 μg mL-1, which was separated by HSCCC. The HSCCC separation was performed with a two-phase solvent system composed of n-butanol-0.1mol/L HCl (1:1, v/v) at a flow rate of 1.2 mL min-1. After a single run, 3 mg hydroxysafflor yellow A (HSYA) with 98% purity, a major component, was separated from 20 mg crude sample Ⅱ. To increase the yield of HSYA and investigate the minor compositions, the sample size of HSCCC separation was enlarged. Crude sample Ⅴ composed of HSYA was obtained. To further increase purity of HSYA and remove the acid which maybe destroyed the natural antioxidants, the Sephadex LH-20 chromatography was employed and eluted by distilled water at the flow rate of 1.5 mL min-1. The separation yielded 184 mg HSYA (1), 10 mg 5,4’-dihydroxyflavone-3,6-di-O-β-D-glucoside-7-O-β-D-glucuronide (2), 3.0 mg chlorogenic acid (3), and 3.2 mg 4-O-β-D-glucosyl-trans-p-coumaric acid (4) from 1 g crude sample Ⅱ of Carthamus tinctorius L florets. The purities of isolated compounds were over 95 %, and their structures were confirmed by MS and NMR. Compound 3 and 4 were found for the first time from this plant. Antioxidant activities assayed in vitro by ABTS radical cation scavenging shown that the SC50 values of four compounds were 44.39±1.62 μg mL-1, 78.13±1.00 μg mL-1, 21.85±1.96 μg mL-1, and 31.44±2.06 μg mL-1, respectively (Fig. 1).

Figure 1. The ABTS radical scavenging activities of the isolated compounds and control sample References

1. Li D; Mündel H. Safflower, Carthamus tinctorius L. Bioversity International, Rome, 1996, p. 8-9. 2. Suleimanov T A, Chem. Nat. Compd. 2004, 40, 13-15. 3. Berthod A; Maryutina T; Spivakov B; Shpigun O; Sutherland I A. Pure Appl. Chem. 2009, 81, 355-387. 4. Ito, Y. J. Chromatogr. A 2005, 1065, 145-168.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-12 Preparative isolation and purification of tanshinones from salvia miltiorrhiza bunge by a novel counter-current chromatography with an upright conical coils

Junling Liang1, Jie Meng1, Shihua Wu*,1

1Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences, Zhejiang University, Hangzhou 310058, P.R. China *Corresponding author. Tel: +86-571-88206287; Fax: +86-571-88206287; E-mail: [email protected]. Key words: counter-current chromatography, conic holder hub, Salvia miltiorrhiza Bunge, Tanshinones

Abstract Counter-current chromatography (CCC) has been playing a critical role in the separation of natural products, which has been recognized as an important source of pharmaceutical agents. Since CCC was first invented in 1970, various apparatus have been developed for higher resolution, such as droplet CCC, high-speed CCC (HSCCC), horizontal flow-through coil planet centrifuge (CPC), and high-performance CCC (HPCCC). In addition, recently researches indicated that spiral coil has better separation efficiency including higher retention of stationary phase, and solutes resolution than other CCC coils， such as helical and toridal coils. However, these coils both are distributed in one- or two-dimensional centrifugal force field except the cross-axis CCC which holds a three-dimensional centrifugal field. This work introduced a novel conical CCC with upright conic coils, which was wound on three identical upright conical holder hubs by head-to-tail and left-hand direction (Fig.1). Compared with helical and spiral CCC, conical CCC holds a special centrifugal force gradient both in axial and radial direction enabling the CCC much higher resolution. And in details, it enables different linear velocity not only in the same layer of column but also between different layers, and large volume of column and sample loop which make higher sample loading capacity. In this work, the novel CCC was successfully applied to isolate and purify danshinones from crude extract of Salvia miltiorrhiza Bunge. As shown in Fig. 2, higher resolution has been obtained compared with traditional CCC apparatus by the solvent system of hexane-ethyl acetate –ethanol-water with the volume ratio of 5:5:7:3. As a result, 4 tanshinones were well resolved from 500 mg to 1g crude samples. (A) 1+2+3

(B)

4 1 2 3 Fig.1 The design principle of upright conic holder hub system on the novel CCC apparatus. Fig.2 Chromatographs of traditional CCC apparatus (TBE-300A, Tauto, Shanghai, China) (A) and the novel Conical apparatus (B). The system was both HEEW (5:5:7:3) and the sample size was 100 mg (A) and 500mg (B) with the rotation speed of 900 rpm (A) and 600rpm (B).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-13 Preparative separation of three flavoniods from the flower buds of daphne genkwa by ph-zone-refining vortex counter-current chromatography

Yong Liang1,2 Ying Ma1 Yoichiro Ito1* 1. Bioseparation Technology Laboratory , Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA 2. School of Chemistry and Environment, South China Normal University, Guangzhou,China Yong Liang address: Phone number: 8620-13539873265, E-mail: [email protected] *Correspondence address: Fax: 301-402-0013; E-mail:[email protected] Keywords: flavonoids, Daphne genkwa, pH-zone-refining vortex counter-current chromatography, High-speed counter-current chromatography, preparation separation

Daphne genkwa Sieb. et Zucc. (Yuanhua in Chinese), a well- known traditional Chinese medicinal herb listed in the Chinese Pharmacopoeia [1], has anti-inflammation, anti-leukemia, and anti-tumor activities [2]. Chemical investigations have disclosed that the flower buds of D. genkwa contain main three flavonoids with anti-inflammatory, anti-leukemia and anti-tumor activities [3]. Modern research requires a large amount of pure apigemin, genkwanin and 3-hydroxy- genkwanin in the crude extract for further pharmacological studies and new drug exploration. pH-zone-refining CCC [4] is generally employed as a large-scale preparative technique for separating ionizable analytes. A new technology of pH-zone-refining vortex CCC is used to separate three flavoniods from the flower

Fig. 1. pH-zone-refining vortex CCC separation of D. genkwa extract. buds of Daphne genkwa. The effect on the vortex CCC column separation using with different kinds of base and concentration in the low phase is discussed, a large amount of three of flavoniods with high purity is obtained. Since methyl tert.-butyl ether– acetonitrile– water (4:1:5) had maximum solubility as well as suitable K values for the sample, it was selected as the two-phase solvent system for the pH-zone- refining vortex CCC separation. The method used in pH-zone-refining vortex CCC was based on D. genkwa characteristic acidity which is determined by the number of phenolic hydroxyl groups and their positions in the molecule. Among those target compounds, apigemin is the most acidic, followed by 3-hydroxy-genkwanin and genkwanin in this order. The apigemin could be eluted with the ammonium hydroxide mobile phase used in the ordinary pH-zone-refining system. However, other two target compounds could not be eluted with ammonium hydroxide in the mobile phase since these weakly acidic phenol groups do not form stable salts with ammonium hydroxide. This problem was overcome using a stronger base, such as NaOH or Na2CO3 as an eluter in the aqueous mobile phase. Therefore, a series of different kinds of bases such as NH3, NaOH and Na2CO3 and their concentrations were tested as an eluter in the aqueous mobile phase. The experiment proved that 10 mM Na2CO3 was the most suitable eluter in the mobile phase combined with 10 mM TFA (trifluoroacetic acid) as a retainer in the stationary phase for separation of apigemin, 3-hydroxy-genkwanin and genkwanin. The reason may be that Na2CO3 is a kind of diacidic base, and it produces two pH changes in the process of its dissociation.

Reference 1. China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, Beijing, China 2010:109 2. Lee, M. Y., Park, B. Y., Kwon,,etc, Int. Immunopharmacol.9(2009) 878–885 3. V.P. Androutsopoulos, K. Ruparelia, R.R.J. Arroo, A.M. Tsatsakis, D.A. Spandidos, Toxicology 264 (2009) 162 4. Yoichiro ito, J. chromatogr. A 1065 (2005) 145–16

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-14 Multiple Dual Mode and trapping molecules for Coomassie Brilliant Blue G-250 purification Nazim MEKAOUI*, Karine FAURE and Alain BERTHOD Institut des Sciences Analytiques, Université de Lyon, CNRS, Villeurbanne, France * E-mail : [email protected] ; Fax : +33 472 448 319 Keywords: Countercurrent chromatography, Batch Chromatography, Continuous Chromatography, Coomassie Brilliant Blue, Multiple Dual Mode

In liquid-liquid partition, and in particular in the case of counter-current chromatography, high loading rates can be achieved without peak distortion (non linearity due to overload). The reason is that compounds with simple solution equilibrium have access to a large stationary phase volume in liquid-liquid contactors compared to adsorption chromatography where stationary phase volume is limited to the solid surface [1].

In countercurrent chromatography (CCC), most of the separations are carried out in batch mode. However, two solutions have already been proposed to perform a separation continuously: The first one uses a real countercurrent motion of two non-miscible phases [2]. However, separations with this approach use specifically designed instruments for countercurrent liquid motions, still not available on the market . The second approach, which is semi-continuous, bypasses the instrumental problem and is based on combining multiple ascending and descending chromatographic steps, first introduced and named by Delannay & al.: Multiple Dual Mode [3].

We previously studied the fundamental conditions to operate a Multiple Dual Mode procedure for binary separations [4]. In the current work, this separation method is applied to the purification of a commercial dye of Coomassie Brilliant Blue G-250 (CBB), which is required a higher purity when used as an indicator of amine content in biological material.

The liquid system heptane/1-butanol/water 2:3:4 (v/v) was found satisfactory to separate crude CBB in three groups of components. Firstly a polar group, partitioning in the aqueous lower phase, then an intermediate one, partitioning well between the aqueous and organic phases, and finally an apolar group preferring the organic phase. The dual-mode way was convenient by injecting the crude CBB in the middle of a two coil analytical CCC instrument. The multi dual-mode purification was performed allowing to get rid of the polar impurities in the aqueous phase at the column tail and of the apolar ones in the organic phase at the column head. The intermediate polarity fraction contained the desired dyes without impurities and trapped inside the CCC column. Working this way with seven descending (or head-to-tail) and eight ascending (or tail-to-head) steps, including an additional injection between each pair of changing mode, a total of 200 mg of purified CBB were trapped inside the CCC column obtained from 1 g of crude CBB in 3 h using less than 250 mL of organic solvents. The purified CBB was finally collected at the end of the experiment by simple extrusion. It gave total satisfaction in testing polyclonal antibodies adsorbed onto a monolithic support.

References

1. A. Berthod, Countercurrent chromatography: the support-free liquid stationary phase, Analytical Chemistry Series, Volume 38, Elsevier, Amsterdam, 2002. 2. R. van den Heuvel, B. Mathews, S. Dubant, I.A. Sutherland, J. Chromatogr. A, 2009, 1216, 4147-4153. 3. E. Delannay, A. Toribio, L. Boudesocque, J. Nuzillard, M. Zèches-Hanrot, E. Dardennes, G. Le Dour, J. Sapi, J.H. Renault, J. Chromatogr. A, 2006, 1127, 45. 4. N. Mekaoui, A. Berthod, J. Chromatogr. A, 2011, 1218, 6061-6071.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-15 Multi-dimension counter-current chromatography for high- throughput analysis of natural products Jie Meng1, Shihua Wu1 1Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences, Zhejiang University, Hangzhou, 310058, P.R China *Corresponding author. Tel: +86-571-88206287; Fax: +86-571-88206287; E-mail: [email protected].

Keywords: Multi-dimension counter-current chromatography; High-throughput; Natural products

ABSTRACT: Counter-current chromatography (CCC), as a unique and important tool for separation and purification of natural products, possesses a peculiar characteristic that is support-free liquid-liquid chromatography, thus it eliminates such complications occurred in common column chromatography as sample loss, tailing of solute peaks and contamination. That is why counter-current chromatography is gaining more and more popularity in modern separation of natural products despite of it’s a little lower resolution than gas chromatography (GC) and high-performance liquid chromatography (HPLC) or other high-resolution column chromatography. Nowadays, resolution, separation time and sample loading capability is what hinder CCC development most seriously, although the CCC methods and related technologies have been improved rapidly due to worldwide researchers constant effort. Natural products, as a very rich source of biologically active compound for new drug, chemical molecular, protein and gene discovery, are needed to be analysis and prepared more quickly. So high-throughput methods and technologies are pivotal for the CCC advancement. This work will provide a 2-dimension (2D) counter-current chromatography design which is illustrated by figure 1. We can realize the shift from single parallel separation to 2D CCC separation by switching the valve II (blue one in the figure) and valve III (green one in the figure) only. So we can add more loading sample due to the increase of the column volume, which can also improve separation performance. Moreover, this design can help us save labor work of assembling these instruments just by switching these two valves.

II

A

III

B

Figure1. the design program for multi-dimension CCC If valve II is in state 1-2 while valve III is in state 1-2 , the three columns are on parallel separation mode; if valve II is in state 1-2 while valve III is in state 1-6 , column A and column C are connected; if valve II is in state 1-6 while valve III is in state 1-6, column A and column B are connected; if valve II is in state 1-6 while valve III is in state 1-2, column C is parallel with column A-B.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-16 Gentle and high-yield preparative separation method of basophils using a rotary-seal-free continuous-flow centrifuge Hiroyuki Shiono1*, Takuya Matsui1, Tadashi Okada1, Hong Miao Chen2, and Yoichiro Ito3 1Department of Physiology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan 2Huaqiao University, Chenghua Nor.Fengze-qu Quanzhou-city Fujian-Sheng CHINA 3 Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, NHLBI, NIH, Bethesda, MD, USA * Fax: +81-561-63-1289, e-mail: [email protected] Keywords: cell density, cell separation, Percoll, basophil, allergy, atopic dermatisis

Mast cells, residing in the peripheral tissues, and basophils, circulating in the peripheral blood, have been considered as primary effector cells in allergic disorders such as anaphylaxis, asthma, hay fever, atopic dermatitis (AD) and urticaria. However, the studies on basophils have been hampered by their small count in peripheral blood and the lack of appropriate animal model. Since the novel role of basophils on chronic allergic inflammation in an animal model has been reported[1, 2], and methods for the purification of basophils with the specific antibody have been develoed[3], studies on basophils recently attracted much attention. However, the negatively selecting magnetic cell separation method for obtaining pure basohil preparations requires prior enrichment steps. These included centrifugation in hydroxyethyl starch, Ficoll or Percoll density centrifugation and counter-current centrifugal elutriation that require skilled techniques, considerable patience and time. In addition the method suffers from the loss of basophils in the procedure of depleting erythrocytes and harvesting leukocytes. Therefore, we have developed a preparative separationl method of basophils using a rotary-seal-free continuous-flow centrifuge to continuously harvestd cells according to their densities. Ten milliliters of citrate-acid-treated blood samples from healthy volunteers and an AD patient were first diluted to hematocrit value of 20%, and directly followed by a novel continuous flow-through cell separation method. Morphology and purity of separated basophils were assessed by May-Grünwald staining of cytospin preparations. A23187 mediated histamine release was analyzed by enzyme immunoassay (EIA). The expression of CD203c antigen, the activation marker of basophils, was measured using flow cytometry before and after activation with anti-IgE. The average concentration of basophils from healthy volunteers was 23% in the density of 1.075 and 64% in the density of 1.080 in three experiments. The total yield of basophils was approximately 3.6 x 105 cells from 10 ml of the peripheral blood. Basophils from an AD patient was also concentrated in the density of 1.075 at 22% and in the density of 1.080 at 58%. Separated basophils from both samples released histamine by A23187-induced degranulation. CD203c surface expression was higher in the AD patient than that in healthy volunteers. After activation with anti-IgE, the increase of CD203c level in healthy volunteers and the AD patient were found in 51% and 81% of basophils, which were similar to the native basophils in whole blood, respectively. The separated basophils preserved IgE receptors. These results suggested that the present method could separate even the sensitive basophils from the peripheral blood of AD patients as functionally viable basophils. We believe that the present method is useful for a preparative and gentle separation of basophils and other functional cells in high yield.

References 1. K. Obata, K. Mukai, Y. Tsujimura, K. Ishiwata, Y. Kawano, Y. Minegishi, N. Watanabe, H. Karasuyama, Blood 110 (2007) 913-920. 2. Karasuyama H., Mukai K., Tsujimura Y., Obata K., Nat. Rev. Immunol. 9 (2009) 9-13. 3. B. F. Gibbs, K. Papenfuss and F. H. Falcone, Clin. Exp. Allergy 38 (2008) 480-485.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-17 Betalain separation from red beet juice (Beta vulgaris L.) by high-performance countercurrent chromatography (HPCCC) in polar high-salt solvent systems Aneta Spórna-Kucab1*, Svetlana Ignatova2, Ian Garrard2, Sławomir Wybraniec1, Cracow University of Technology, Faculty of Chemical Engineering and Technology, Institute C-1, Department of Analytical Chemistry, Cracow, ul. Warszawska 24, Poland 2 Brunel University, Brunel Institute for Bioengineering, Uxbridge, West London, UK *[email protected] Keywords: countercurrent chromatography, betalains, decarboxylated betacyanins, betanin.

Growing interest in the potential anticarcinogenic health benefits of betalains (betaxanthins and betacyanins) by consumers has created an increased interest in red beet (Beta vulgaris L.) as a low-cost, natural source of these pigments (1). The application of betalains is restricted due to their low stability in certain physicochemical conditions (e.g. oxygen, light, temperature etc.). Decarboxylated and dehydrogenated betalains usually are more stable than their corresponding betalains (2). The low stability of betalains generates some problems with their isolation. So far, the separation of complex mixtures has been realized mainly by HPLC (3). However, the introduction of high-speed countercurrent chromatography (HSCCC) generated new opportunities for obtaining larger amounts of pure pigments whereby the betalains separation was performed mainly in ion-pair solvent systems (4).

In this study, two mixtures of betalains: mixture 1 (betanin, decarboxy-derivatives and 14,15-dehydro-betanin) and mixture 2 (decarboxy-/dehydro-derivatives) were separated. The mixtures were obtained during thermal treatment of red beet juice for 30 min (mixture 1) and 60 min (mixture 2) at 85°C. In the experiment, betalains (mixtures 1, 2) were separated for the first time with high-performance countercurrent chromatography (HPCCC). Separation of the betalains was performed in very polar, high-salt solvent systems composed of:

1-PrOH – ACN - (NH4)2SO4 (satd. soln) - water (v/v/v/v, 1:0.5:1.2:1) – system I, EtOH - ACN - 1-PrOH -

(NH4)2SO4 (satd. soln.) – water (v/v/v/v/v, 0.5:0.5:0.5:1.2:1) – system II, and EtOH - 1-BuOH - ACN -

(NH4)2SO4 (satd. soln.) - water (v/v/v/v/v, 0.5:0.5:0.5:1.2:1) – system III in ‘tail-to-head mode’. The separation of decarboxy-/dehydro-betalains was performed for the first time. The best results were obtained for mixture 1 in systems I and II in which separation of 2,17-bidecarboxy-betanin, 2-decarboxy-betanin and neobetanin was very effective. In contrast, the separation of betanin from 17-decarboxy-betanin was less effective. These compounds eluted during the elution extrusion process. The application of these solvent systems to the separation of the mixture 2 enabled resolution of 17-decarboxy-neobetanin (systems I-III), 2,15,17-tridecarboxy-2,3-dehydro-neobetanin (systems I, III), and 2,17-bidecarboxy-2,3-dehydro-neobetanin (systems III) with high efficiency.

References

(1) Strack, D.; Vogt, T.; Schliemann, W. Recent advances in betalain research. Phytochemistry 2003, 62, 247-269.

(2) Herbach, K., M.; Stintzing, F., C.; Carle, R. Betalain Stability and Degradation - Structural and Chromatic Aspects. J. Food Sci. 2006, 71, R41-R50.

(3) Wybraniec, S. Effect of tetraalkylammonium salts on retention of betacyanins and decarboxylated betacyanins in ion-pair reversed-phase high-performance liquid chromatography. J. Chromatogr. A 2006, 1127, 70-75.

(4) Wybraniec, S.; Stalica, P.; Jerz, G.; Klose, B.; Gebers, N.; Winterhalter, P.; Spórna, A.; Szaleniec, M.; Mizrahi, Y. Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography. J. Chromatogr. A 2009, 1216, 6890-6899. 7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-18 Separation of decarboxy-/dehydro-betalains by high-performance countercurrent chromatography in ion-pair solvent systems. Aneta Spórna-Kucab1*, Svetlana Ignatova2, Ian Garrard2, Sławomir Wybraniec1 Cracow University of Technology, Faculty of Chemical Engineering and Technology, Institute C-1, Department of Analytical Chemistry, Cracow, ul. Warszawska 24, Poland 2 Brunel University, Brunel Institute for Bioengineering, Uxbridge, West London, UK *[email protected] Keywords: countercurrent chromatography, betalains, Beta vulgaris L., ion-pair solvent systems.

Beta vulgaris L. is a source of natural food colorants – betalains (betacyanins and betaxanthins).The main betacyanin is betanin, a compound which shows poor stability under certain physicochemical conditions (low pH, temperature, light, etc.) (1, 2). The main pathways of betanin degradation are: oxidation, hydrolization, deglucosylation, decarboxylation, dehydrogenation and deglucosylation. The pigments have potential health benefits and non-toxic features, so they are very attractive compounds for consumers and scientists. Research on the different structures is possible after isolation of the pigments. So far, the separation of betalains has been performed mainly by HPLC (3). Recently high-speed countercurrent chromatography (HSCCC) was used to separate betalains and their acylated derivatives (4).

In this study, ion-pair high-performance countercurrent chromatography (HPCCC) was used for the first time to separate betalains and their decarboxy-/dehydro-derivatives. In the experiment, two mixtures (1, 2) were used. Mixture 1 was a product of the red beet juice heated at 85°C for 30 min and mixture 2 for 60 min. The HPCCC process was carried out in reversed phase mode with five solvent systems composed of tert-butyl-methyl-ether (TBME) – butanol (Bu-OH) – acetonitrile (ACN) – water (acidified with 0.4%, 0.7% and 1% of TFA or HFBA – the ion-pair reagents). The flow rate of the mobile phase was 0.25 ml/min and the column rotation speed was 2049 rpm (20 °C). The results of the separation depended on the type of acid used as an additive and its concentration. Moreover, the acid concentration influenced the stationary phase retention, causing its decrease at higher concentrations. The application of the ion-pair solvent systems resulted in betalain chromatographic profiles that were significantly different from the profiles observed in C18-HPLC, due to the formation of ion-pairs between the non-dehydrogenated betalains and anions of the acids. The study confirmed that the ion-pair solvent system with HFBA is more effective than with TFA (4) and the best results were obtained for the solvent systems with 0.7% HFBA. This ion-pair solvent system was more effective for the dehydrogenated betacyanins in mixture 2 (17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin, 2,15,17-tridecarboxy-neobetanin, and 2-decarboxy-neobeta-nin) than for the more polar pigments in the mixture 1 (betanin, 17-decarboxy-betanin, 2-decarboxy-betanin, 2,17-bidecarboxy-betanin, and neobetanin).

References:

(1) Stintzing, F., C.; Carle, R. Betalains – emerging prospects for food scientists. Trends Food Sci. Tech. 2007, 18, 514-525.

(2) Herbach, K., M.; Stintzing, F., C.; Carle, R. Betalain Stability and Degradation - Structural and Chromatic Aspects. J. Food Sci. 2006, 71, R41-R50.

(3) Wybraniec, S. A method for identification of diastereomers of 2-decarboxy-betacyanins and 2,17-bidecarboxy-betacyanins in reversed-phase HPLC. Anal. Bioanal. Chem. 2007, 389, 1611-1621.

(4) Wybraniec, S.; Stalica, P.; Jerz, G.; Klose, B.; Gebers, N.; Winterhalter, P.; Spórna, A.; Szaleniec, M.; Mizrahi, Y. Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography. J. Chromatogr. A 2009, 1216, 6890-6899.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-19 Devalopment of new pattern PTFE column of HSCCC Wang Gao-hong1,2, Chen Xiao-Fen1,2, Huang Xin-Yi1, Yun Liu1,2, Di Duo-Long1* 1 Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000 2 Graduate University of the Chinese Academy of Sciences, Beijing 100049 *Corresponding author fax: 0931-4968094; e-mail: [email protected] Keywords: Column; PTFE; Flavonoid; HSCCC Based on the multilayer coil polytetrafluroethylene (PTFE) column as separation column, the separation and purification of chemical components from complex matrix was carryed out using suitable two-phase solvent system on HSCCC. The separation mechanism of conventional HSCCC relegates to a continuous liquid–liquid partition chromatography with no solid support and can eliminate irreversible adsorption of samples on solid support. However, it is difficult to separate compounds which possess similar structure and distribution coefficient in complex samples depended simply on single liquid–liquid partition model. In order to improve separation efficiency on HSCCC to compounds which possess similar structure and distribution coefficient in complex samples, a new pattern of PTFE column would be developed. Firstly, in order to increase inner-surface roughness and enlarge surface area, sodium naphthalene solution was employed for pre-treating the inner-surface of PTFE column. Secondly, PTFE column was filled with dopamine solution and the dopamine self-assembly was performanced on the inner-surface of PTFE column. The modified process is shown in figure 1. As observed in SEM graphs (figure 2), the inner-surface of untreated PTFE column was quite smooth (see figure 2-a), while that of treated PTFE column was rather rough (see figure2-b). Poly-dopamine particles could be found on the inner-surface of coated PTFE column (see figure 2-c). The treated PTFE column was used for separation of three flavonoid compounds (quercitin, kaempferol and isorhamnetin). The result showed that this new pattern PTFE column could visibly improve separation efficiency of three flavonoid compounds above-mentioned.

Figure 1.The modified process of HSCCC column: (a) untreated, (b) treated, (c) coated.

Figure 2. SEM graphs of PTFE: (a) untreated, (b) treated, (c) coated.

Acknowledgements: Authors gratefully acknowledge the financial support by the ‘Hundred Talents Program’ of the Chinese Academy of Sciences (CAS), the National Natural

Sciences Foundation of China (NSFC No. 20974116, 21175142).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-20 Multiple dual mode centrifugal partition chromatography as an efficient method for the purification of 4-phenylcoumarins from Mesua elegans.

Chan Gomathi1, Joel Boustie2, Grégoire Audo*3, Nor Hadiani Ismail4, Céline Le Quémener3, Khalijah Awang1 1Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Laboratoire de Pharmacognosie et de Mycologie, Faculté de Pharmacie, PNSCM Team, UMR 6226, Univ Rennes 1, 2 Av. Du Pr. Léon Bernard-35043 Rennes, Cédex, France 3Armen instrument, ZI Kermelin, 16 rue Ampère, 56890 Saint Avé, France. [email protected] 4Faculty of Applied Sciences, Universiti Teknologi Mara, 40450 Shah Alam, Selangor, Malaysia. Keywords: Multi dual mode, phenylcoumarins, automation

Centrifugal Partition Chromatography (CPC) was applied to fractionate a hexane enriched extract of Mesua elegans (Clusiaceae). A direct comparison between classical isocratic mode and multi-dual mode with a two-phase solvent system composed of heptane-ethyl acetate-acetonitrile-water (9:1:9:1, v/v/v/v) was performed. Optimization of injection capacity was done with both method by injecting successively 50, 150, 300 and 500 mg of crude extract. Two 4-phenylcoumarins; mammea A/BA and mesuagenin C (Figure 1), were obtained from the extract with 280 nm HPLC purity of 93.2 % and 94.3 % in 500 mg injection in multi-dual mode (Table 1), compare to 82.3% and 79.4% in isocratic mode (Table 2). Structures of these two compounds were identified by 1H NMR, 13C NMR and LC-MS. Multi-dual mode allows in these case to increase the injection capacity without decreasing purity of target compounds.

Table 1. 280 nm HPLC purity of Mammea A/BA Table 2. 280 nm HPLC purity of Mammea A/BA and and Mesuagenin C after multi dual mode CPC Mesuagenin C after isocratic CPC mode.

Multi-dual mode Isocratic descending mode Mass Mass Mammea A/BA1 Mesuagenin C2 Mammea A/BA1 Mesuagenin C2 Injected Injected 50 mg 99.7% 100% 50 mg 98.5% 98.1% 150 mg 97.6% 95.5% 150 mg 96.2% 94.6% 300 mg 93.0% 96.6% 300 mg 84.2% 91.1% 500 mg 93.2% 94.3% 500 mg 82.3% 79.4%

1 2

Figure 1. mammea A/BA (1) and mesuagenin C (2)

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-21 Preparative separation of two subsidiary colors of FD&C yellow NO. 5 (tartrazine) by spiral high-speed counter-current chromatography Adrian Weisza*, Jose A. Roquea, Eugene P. Mazzolab, Yoichiro Itoc a*Office of Cosmetics and Colors, and bOffice of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Pkwy, College Park, MD 20740, USA cBioseparation Technology Lab., Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA *fax: +1 301-436-2961; [email protected] Keywords: Sulfonated dyes, Spiral HSCCC, Ultra polar biphasic solvent system FD&C Yellow No. 5 (Y5, Tartrazine, Colour Index No. 19140) is a color additive used in food, drugs, and cosmetics in the U.S. (1). It is manufactured by condensing phenylhydrazine-p-sulfonic acid with an oxalacetic ester, coupling the product with diazotized sulfanilic acid, and then hydrolyzing the ester with sodium hydroxide (2). The reaction produces mainly 1 (Fig. 1, azo form) accompanied by a variety of organic impurities, which include intermediates, side-reaction products, and subsidiary colors. These impurities are carried over during the manufacturing process and can be found in the final product. Y5 is batch-certified by the U.S. Food and Drug Administration (FDA) to ensure compliance with specifications in the Code of Federal Regulations (CFR). Among the specifications is a limit of 1% for each of two polysulfonated subsidiary colors labeled “Pk5” and “Pk7” in Figure 2.

Figure 1 Main component of FD&C Yellow No. 5

Figure 2 HPLC chromatogram of the sample of Y5 used in the present work. Currently, the determination of these subsidiary colors is performed by an indirect method because FDA does not have reference materials for Pk5 and Pk7. The present work describes the use of spiral high speed counter-current chromatography (HSCCC) to separate preparative quantities of Pk5 and Pk7 from a sample of Y5 that contained ~1.24% Pk5 and ~0.73% Pk7 (3). Due to the high polarity of the compounds to be separated, spiral HSCCC was used in combination with an organic/high-ionic-strength aqueous two-phase solvent system (4). The instrument used was a CCC-1000 Pharma-Tech J-type HSCCC fitted with three spiral-tube assembly coils (CC Biotech LLC) resulting in a column with total capacity of 225 ml. The solvent system 1-butanol-ethanol-saturated ammonium sulfate-water (1.6:0.4:1:1, v/v) was chosen based on the graphic optimization of the partition coefficients recently described (4). Two steps were needed to separate Pk5 and Pk7 from samples (50-300 mg) of Y5. In the first step, Pk5 was separated by using the aqueous lower phase as the mobile phase and Pk7, which is less polar than Pk5, was retained in the organic stationary phase. In the second step, Pk7 was separated by using the organic phase as the mobile phase. The separated components were identified by NMR and high-resolution MS techniques. The separated materials will be used for qualitative and quantitative confirmatory analyses in order to enforce the CFR specifications for Pk5 and Pk7 in Y5. References 1. Code of Federal Regulations, 21 CFR 74.705, U.S. Government Printing Office, Washington, DC, 2011. 2. K.A. Freeman, J.H. Jones, C. Graichen, J. Assoc. Off. Agr. Chemists 33 (1950) 937. 3. Y. Ito, R. Clary, J. Powell, M. Knight, T.M. Finn, J. Liq. Chromatogr. Rel. Technol. 31 (2008) 1346. 4. Y. Zeng, G. Liu, Y. Ma, X. Chen, Y. Ito, J. Liq. Chromatogr. Rel. Technol. (2012) in press.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-22 Novel sample pretreatment method for increasing the preparative scale of pH-zone refining counter-current chromatography Yi Yang, Dongyu Gu, Qibien Chen, Abulimiti Yili, Haji Akber Aisa* Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China *Corresponding author: Haji Akber Aisa, Tel: +86 991 3835679, Fax: +86 991 3838957, E-mail: [email protected]

Keywords: crude sample preparation, yield, pH-zone refining counter-current chromatography pH-zone refining counter-current chromatography (CCC) is classic large-scale preparative technique for separating ionizable compounds [1,2]. The typical sample size separated by high-speed counter-current chromatography (HSCCC) instrument with 320 ml column capacity may range from 0.5 to 5 g [3]. As we known, the selection of solvent system is the most important step for pH-zone-refining CCC separation [3]. For an acidic analyte, when pH of two-phase solvent system is around 2, Kacid should be much bigger than 1. And for a basic analyte, when pH of solvent system is about 10-12, Kbase shold be much bigger than 1 [3]. Usually, after selection of two-phase solvent system, pH-zone-refining CCC sparation procedure will begin imediataly. However, if the selected two-phase solvent system without acid or base in the lower phase is used for extraction of target compound, most of this compound would be enriched in the upper phase because of very large K value of targer compound in this solvent system. So the content of target compound would be increased. If the same crude sample mount was loaded into CCC column, the yeild of target compound will increased. Base on this idea, a novel method for crude sample pretreatment for pH-zone-refining CCC sparation was established in the present study. The separation of alkaloids with antitumor activity in the seeds of Sophora alopecuroides, a traditional medicine, was presented as example of our method [4]. Two-phase solvent system composed of methyl tert-butyl ether-acetonitrile-water (2:2:3, v/v), where triethylamine (10mM) was added to the organic phase as a retainer and hydrochloric acid (10 mM) to the Figure 1. Chromatograms of alkaloids separation aqueous phase as an eluter, was selected and used in the separation of pH-zone-refining CCC. Before CCC from crude sample I (A) and II (B) of Sophora separation, 20 g total alkaloids (crude sample I) was alopecuroides seeds obtained by pH-zone extrated by this solvent system, where triethylamine (10 refining CCC. mM) was added to the organic phase. And 10 g crude sample II was obtained. 2 g crude sample I and II were separated by pH-zone-refining CCC under the same conditions, respectively. And 98 mg sophoramin and 371 mg sophoearpin were isolated from crude sample I (Fig. 1A). And 169 mg sophoramin and 696 mg sophoearpin were isolated from crude sample II (Fig. 1B). The purities of products were similar and over 95%. And when the 1 g crude sample II from 2 g crude sample I was separated by CCC. The retention of stationary phase was remarkable increased. And similar mounts of two compounds were obtained. Thus, the target compounds were efficiently enriched by this sample pretreatment method in the present study. Although one extraction step was added in the procedure of crude sample preparation, the yields of products of pH-zone-refining CCC were increased to approximately 2 times. References

1. Weisz, A.; Scher, A. L.; Shinomiya, K.; Fales, H. M.; Ito, Y. J. Am. Chem. Soc. 1994, 116, 704-708. 2. Ito, Y; Ma, Y. J. Chromatogr. A 1996, 753, 1-36. 3. Ito, Y. J. Chromatogr. A 2005, 1065, 145-168. 4. Zhang L; Wang T; Wen X; Wei Y; Peng X; Li H, Wei L. Eur. J. Pharmacol. 2007, 563, 69-76.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-23 Solvent gradient counter-current chromatography purification of podophyllotoxins from Dysosma versipellis (hance) Zhi Yang1, Shihua Wu1*

1Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life Sciences Zhejiang University, Hangzhou 310058, China *Corresponding author. Tel: +86-571-88206287; Fax: +86-571-88206287; E-mail: [email protected] Keywords: solvent gradient counter-current chromatography; linear solvent gradient ccc separation; step-gradient ccc separation; podophyllotoxins; Abstract: Stepwise elution modes have been frequently applied in the CCC separation to isolate compounds with a large range of polarities. During the preparation of Dysosma versipellis (Hance) by HSCCC, all the compounds can be separated in a short time in hexane-ethyl acetate-methanol-water (4646) system, but compounds 1 （4′-demethylpodophyllotoxin）and 2（β-peltatin-5-O-β-D-glucopyranoside） cannot be separated soundly. In hexane-ethyl acetate-methanol-water (4637) system, compounds 1 and 2 can be separated much better, while it takes a much longer time. When a stepwise elution mode is applied, sound separation and short time are obtained. In this work we applied two stepwise elution modes--Linear solvent gradient CCC separation and Step-gradient CCC separation. And Step-gradient CCC separation proved more effective in this work. In linear solvent gradient CCC separation, the changes of the system are more mitigatory and the reduction of the stationary phase exists during the whole changing process. While in the step-gradient CCC separation, new hydrodynamic equilibrium can be established more quickly. And we found that the reduction of the stationary phase was the same in the two modes. The two modes are both effective methods for quick separations. According to different separation, one more effective mode should be selected.

R1 4' MeO 3' OMe 5' 6' 2' O 1' 1 8 2a 9 7 O 2 O 3 6 10 O 3a 4 5 R2 R3

R1 R2 R3 4′-demethylpodophyllotoxin (1): -OH -OH -H β-peltatin-Glu (2): -OCH3 -H -Glu β-peltatin (3): -OCH3 -H -OH podophyllotoxin (4): -OCH3 -OH -H Figure 1.Stuctures of podophyllotocins separated from dysosma versipellis (hance)

Figure2.The elution curves of the preparative CCC. 1. 4′-demethylpodophyllotoxin 2. β-peltatin-5-O-β-D-glucopyranoside 3. β-peltatin 4. podophyllotoxin. CCC separation conditions: A: Stationary phase, the upper phase of HEMWat (4646). Mobile phase, the lower phase of HEMWat (4646), Retention of the stationary phase, 59.26%. B: A Stationary phase, the upper phase of HEMWat (4637). Mobile phase, the lower phase of HEMWat (4637), Retention of the stationary phase, 61.11%. C: Stationary phase, the upper phase of HEMWat (4637). Mobile phase, 0-120 min, the lower phase of HEMWat (4637), 120-255 min, the lower phase of HEMWat (4637) from 100% to 0 and the lower phase of HEMWat (4646) from 0 to 100%, after 255 min , the lower phase of HEMWat (4646). D: Stationary phase, the upper phase of HEMWat (4637). Mobile phase, 0-120 min, the lower phase of he HEMWat (4637), after 120 min, the lower phase of HEMWat (4646). Flow rate, 2 mL/min. Rotation speed, 900 rpm. Detection, 254 nm. Sample size, 77 mg of the fraction two was prepared by dissolving the crude in a solution composed of the upper and lower phases (1:1, v/v, 4 mL of the total volume)..

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-24 Preparative separation of isoquiinoline alkaloids from coptis japonica by high-speed counter-current chromatography Guijae Yooa, Tae Bum Kima, Heejung Yanga, Jin-Ho parkb, Young Choong Kima,b, Sang Hyun Sunga,b,* a College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea b Institute for Life Science, Elcom science Co. Ltd., Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea *Corresponding author: [email protected]

Keywords: Coptis japonica Makino, Alkaloids

High-speed countercurrent chromatography (HSCCC) was applied to separate five isoquinoline alkaloids, palmatine (1), berberine (2), epiberberine (3), coptisine (4) and columbamine (5), isolated from BuOH extract of Coptis japonica Makino (Ranunculaceae). The two phase solvent system comprising chloroform–methanol–0.2 M hydrochloric acid (4:1.5:2, v/v/v) was employed to HSCCC and the flow rate of mobile phase was optimized at 1.5 ml/min and rotated at 800 rpm. The monitoring of HSCCC peak fractions was performed by combining effluent line of the HSCCC apparatus to UV detector (280 nm). Isolated compounds were elucidated by ESI-MS and several NMR techniques including 1D and 2D NMR spectroscopic methods.

Parent ion Yield # fraction Compound Figure 2. HPLC-ESI-MS spectrum of the five isoquinoline ([M]+) (mg) alkaloids in Coptis japonica n-BuOH extracts 1 Palmatine 352.24 9

2 Berberine 336.26 6

3 Epiberberine 336.24 6

4 Coptisine 320.27 4

5 Coptisine 320.28 4

6 Columbamine 338.23 4 Compound R1 R2 R3 R4

Figure 1. HSCCC chromatography of the Coptis Palmatine CH3 CH3 CH3 CH3 japonica n-BuOH extracts Berberine CH3 CH3 —CH2—

References Epiberberine —CH2— CH3 CH3

Coptisine —CH2— —CH2— 1. Yang, F; Zhang, T; Zhang, R; Ito, Y. J. Chromatogr. A. 1998, 829, 137. Columbamine CH3 CH3 CH3 H 2. Yoon, K. D.; Chin, Y. W.; Yang, M. H.; Kim, J. W. Figure 3. The chemical structures of isoquinoline alkaloids in the Food Chem. 2011, 129, 679. n-BuOH extract of Coptis japonica

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-25 Screening for anti-diabetes fraction in the leaves of Olea europaea L. by HPCCC Zhang Jia1,3, Huang Xin-Yi1, Pei Dong1, Chen Xiao-Fen1,2, Di Duo-Long1,3* 1 Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000 2 Graduate University of the Chinese Academy of Sciences, Beijing 100049 3 Department of Pharmacy，Gansu College of Traditional Chinese Medicine，Lanzhou 730000 *Corresponding author fax: 0931-4968094; e-mail: [email protected] Keywords: Olea europaea L; anti-diabete; HPCCC Modern pharmacological studies have shown that the leaves of Olea europaea L. have variety of pharmacological activities, such as anti-bacterial, anti-inflammatory, antioxidant. However, there are few reports about the anti-diabetic activity of Olea europaea L. leaves. In this work, different fractions named A-F were prepared by macroporous adsorption resins from ethanol extracts of the Olea europaea L. leaves. The α-amylase and non-enzymatic glycation system were employed to evaluate the anti-diabetes activities. The results was found that fraction D shown potential anti-diabetic activity. Subsequently, screening and preparative separation of active components from the leaves of Olea europaea L. was performanced by HPCCC (High Performance Counter-Current Chromatography). The separation was achieved on a DE Spectrum HPCCC under analytical condition with solvent system ethyl acetate–water (1:1, v/v). The upper phase as stationary phase and mobile phase was pumped at a flow rate of 0.8 mL/min at 1500 rpm. Six constituents were obtained (See figure 1), and the anti-diabetes activity was evaluated using above mentioned method. The result was found that four constituents were screened out to possess potential activities. Owing to the numbered 1 constituent in figure 1 was admixture , further separation was performanced as follows: butanol–water-acetic acid (1:1:0.1, v/v) was selected as solvent system, the upper phase was stationary phase and mobile phase was pumped at a flow rate of 0.5 mL/min at 1500 rpm, 8 fractions were obtained (See figure 2). The result was found that 3 constituents in figure 2 from 1 constituent in figure 1 were screened out to possess potential activities.

Figure 1. CCC chromatogram of fraction D Figure 2. CCC chromatogram of constituent 1

Acknowledgements: Authors gratefully acknowledge the financial support by the ‘Hundred Talents Program’ of the Chinese Academy of Sciences (CAS), the National Natural Sciences Foundation of China (NSFC No. 20974116, 21175142).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-26 Flow separation of cells in counter-current chromatography (CCC) centrifuges Tian Han, Remco van den Heuvel, Ian A Sutherland and Derek Fisher Advanced Bioprocessing Centre, Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UK *Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, United Kingdom. Fax: +44 (0) 1895 274608. E-mail: [email protected] Keywords: Flow separation, cell separation, CCC centrifuge Stem cell therapy requires the separation of differentiated from undifferentiated stem cells. The aim of our research is to investigate and develop new cell separation methods based on flow separation in counter-current chromatog-raphy (CCC) centrifuges that could be applied to stem cell separations. There have been few reports of cell separations by CCC. Ito et al separated red blood cells (RBCs) using a non synchronous CCC centrifuge in a single phase of physiological saline (1) and an aqueous two phase system (2). To understand the behaviour of cells in CCC centrifuges we have started with a study of the effect of flow rate and rotational speed (fluctuating g force) on the behaviour of a model system of sheep and human RBCs, flowing in a single phase of physiological saline. In contrast to Ito et al (1), initially we have used a synchronous CCC centrifuge: a Milli-CCC J-type centrifuge (3) with an 8.2 mL, 1 mm ID, multilayer column, filled with an isotonic NaCl-phosphate buffer (pH 7.4) (PBS). RBCs in saline were injected and 1ml fractions collected. Inline monitoring at 405nm detected cells and haemoglobin (Hb). Free Hb and intact cells containing Hb were distinguished by off line analysis in a plate reader after and before centrifugation (3000RPM, 10min) respectively. Depending on the flow rate and the rotational speed RBCs were sedimented and retained in the column or eluted in the column volume. For a particular rotational speed there was a minimum flow rate which caused all the cells to be retained and a maximum flow rate when all cells were eluted. Between these two flow rates some cells were retained and some eluted. When the flow rate was increased from the minimum flow rate retained cells could be eluted from the column (Fig 1). Human RBCs had a higher min flow rate than the sheep RBCs, indicating that human RBCs are retained easier than sheep RBCs. But, human RBCs also have a lower max flow rate, indicating that human RBCs are also pushed out easier. This difference has been used to develop a separation method using a flow gradient (Fig.2). Interestingly the order of RBC elution (human then sheep) is opposite to that obtained by Ito et al (1) using a nonsynchronous CCC indicating a difference in the separation mechanisms.

B

A

Fig.1 Retention and elution of sheep RBCs in CCC coil. Sheep RBCs were injected into CCC coil filled with PBS at 900rpm and 0.8 ml min (close to min flow rate). Free Hb eluted at the column volume (Peak A) and RBCs were retained. Decreasing speed to 500rpm and increasing flow to 5ml/min (after arrow point) eluted the RBCs (Peak B). A405 before and after centrifugation distinguishes intact cells from free Hb.

Sheep RBCs

Human RBCs

Fig.2Cell separation. A mixture of sheep and human RBCs was retained in the coil at 800rpm, 0.5ml/min and then eluted differentially by increasing flow to 10ml/min in intervals of 0.1ml/min each 2min. The effects of coil internal diameter, length, and rotation direction on the behaviour of RBCs in the coil will also be reported.

References 1.Ito Y, Carmeci P & Sutherland, IA (1979) Anal. Biochem. 94, 249-252 2. Sutherland, IA & Ito Y (1980) Anal. Biochem. 108, 367-373 3. Janaway L, Hawes D, Ignatova S, Wood P & Sutherland IA (2003) J. Liq. Chrom.R.T., 26, 9-10, 1345-1354.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-27 Comparative study of three modifications of the controlled-cycle liquid-liquid chromatography device Andrey A. Erastov, Yulya A. Zakhodjaeva, Andrey A. Voshkin, Artak E. Kostanyan* Kurnakov Institute of General & Inorganic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 31, Moscow 119991, Russia fax +495 9554834, e-mail: [email protected] Keywords: counter-current chromatography; controlled-cycle operation In this report, we attempt to compare the three versions of the controlled-cycle liquid-liquid chromatography (CLC) device evaluating their similarities and benefits by using a model sample system containing caffeine and coumarin. The CLC apparatus consists of a series of multistage columns connected in the form of a coil, and a pulsation-cycling flow system for regulated discrete supply of the mobile phase as repetitive impulses to the apparatus (1,2). The columns are divided into stages by equally spaced horizontal perforated plates. The pulsation-cycling flow system presents a piston pump with adjustable piston stroke length and PC regulated frequency and piston velocity. An operating cycle of the CLC apparatus consists of two individually timed periods: 1 - flow (contact) period, during which a specified portion of mobile phase is displaced downward being dispersed in the stationary phase in each stage of the columns; 2 - settling period, during which the droplets of the heavy mobile phase coalesce in the stages forming layers at the bottom of each stage. Experiments were carried out with three constructions of the columns (designed and fabricated in our Institute): 1 – column of 6.4 mm internal diameter FEP tubing divided into 26 equal stages by perforated plates (from PTFE sheet, 3 mm thick with 0.25 mm diameter holes) at 35 mm intervals; 2 – column of 8 mm internal diameter FEP tubing divided into 52 stages by stainless steel plates (0.3 mm thick with 0.1 mm diameter holes) at 5 mm intervals; 3 - column of 11 mm internal diameter FEP tubing divided into 21 stages by PTFE perforated plates (2 mm thick with 0.25 mm diameter holes) at 15 mm intervals. Column 3 is equipped with agitators, located in each stage and operating in the cyclic (periodic) mode. The chromatographic peaks of caffeine (KD =0.58) and coumarin (KD =1.65) obtained by using column 3 with the hexane-isopropanol-water biphasic system (volume ratio 1:1:1) are shown in Fig. 1. The units on the horizontal axis (i) represent the number of mobile phase transfers.

Figure 1. Experimental chromatographic peaks of caffeine (KD =0.58) and coumarin (KD =1.65) obtained on the column 3 (with agitators). The advantage of the column3 (with agitators) over the first two versions is the possibility to adjust the contact time for each specific separation process. This is particularly important when the interphase mass transfer is accompanied by chemical reactions, as for example, in the processes of metal separation. Of all three investigated columns, by using hexane-isopropanol-water biphasic system column 3 demonstrated the highest efficiency (measured with the number of theoretical plates).

References

1. A.E. Kostanyan, A.A. Voshkin, N.V. Kodin, J. Chromatogr. A 1218 (2011) 6135. 2. A.E. Kostanyan, A.A. Voshkin, N.V. Kodin, Theor. Found. Chem. Eng. 45 (5) (2011) 779.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-28 Targeted analysis of natural products by K-based countercurrent separation Feng Qiu1, J. Brent Friesen1,2, James B. McAlpine1, David C. Lankin1, and Guido F. Pauli1,*

1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA; 2Physical Sciences Department, Rosary College of Arts and Sciences, Dominican University, River Forest, IL 60305, USA. * [email protected] fax +1 312 355 2693 Keywords: Ginkgo biloba, Camellia sinensis, countercurrent separation, qNMR

Countercurrent separation (CS) is based on the differences in partition coefficients (K) of analytes distributed between a liquid stationary phase and a liquid mobile phase. The K value is not only an important theoretical parameter in CS, but also has much practical value. In this study, K values were used to predict the CS profiles of analytes, assist in the selection and optimization of CS solvent systems, and guide targeted separation. The present study developed a quantitative 1H NMR (qHNMR)-based approach for the simultaneous measurement of K values of five Ginkgo biloba terpene lactones directly from crude plant extracts. Furthermore, two-dimensional selectivity provided by a pair of orthogonal CS solvent systems was introduced in order to enhance the resolution of these difficult-to-separate natural product congeners. Using the K values in optimized orthogonal solvent systems, a “K map” was created that guided locating the target compounds in series of CS fractions (see Figure 1). Consequently, all five terpene lactones could be separated, yielding isolates with qHNMR purities of > 95%, directly from Ginkgo biloba leaf extracts.

The separation was further evaluated by offline qHNMR analysis of CS fractions (offline CS-NMR) associated with Gaussian curve fitting. The Gaussian nature of CS elution profiles results from CS theory and is supported by the experimental NMR-hyphenated CS data. NMR-guided and targeted K-based countercurrent separation was also applied to develop a separation scheme for green tea (Camellia sinensis) catechins.

The structural selectivity of NMR and the adjustable chromatographic selectivity of CS provide a highly versatile platform for the targeted analysis of natural products and other complex materials, utilizing the partition coefficients (K) as an important predictive and accessible parameter.

Figure 1. Visualization of how target compounds can be separated and isolated by using orthogonality-enhanced CS in a pair of specially designed SSs.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-29 Characterisation of quaternary Heptane-Water based phase systems across the polarity range for Counter-current Chromatography R.N.A.M. van den Heuvel1, G. Harris2, N. Douillet3, C. Thickitt3, E.A. van den Heuvel1, S. Ignatova1,* 1,* Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UK, Tel: +44(0)1895 266911, Fax:+44(0)1895 274608, [email protected] 2 Dynamic Extractions Inc, NJ 08852, USA 3 GlaxoSmithKline, Pharmaceuticals R&D Facility, Stevenage, SG1 2NY, UK Keywords: countercurrent chromatography, phase system characterisation, polarity range The recent development of reliable and scalable instrumentation for counter-current chromatography (CCC) coupled with new and unique modes of operation have increased the potential mainstream applicability of this technique. A CCC column simply consists of a biphasic mixture of liquids with the separation being determined by the solute distribution ratio between the two phases. The number of potential columns (solvent systems) is almost infinite. However, solvent system selection, equivalent to column selection for HPLC and SFC, still remains a domain of those skilled in the art. Commonly used solvent systems for CCC are composed of Heptane, Ethyl acetate, Methanol, and Water (HEMWat or Arizona) in regularly changing proportions, creating a series from non-polar to polar (1,2). On-demand preparation of this series using conventional HPLC equipment has recently been demonstrated allowing the automation of the solvent system scouting process (3,4). This has enabled rapid development of separations for solutes compatible with the HEMWat series. If a sample is not HEMWat compatible there are limited examples of alternative solvent system tables. To date there are at least two approaches described in the literature for developing solvent system tables. In one approach, demonstrated by HEMWat, the overall polarity difference between phases remains the same. This type of systems covers a wide range of polarity. Another approach, for example a Heptane, Toluene, Acetone and Water system (HTAW) (5), is to fix the content of one of the modifiers (eg Acetone in the HTAW system) while ratios of all other solvents change. This covers a much narrower polarity range. Although the HTAW system has been in use for some time, it has never been fully characterised. The aim of this study was to create alternative solvent system tables covering different ranges of polarity and solubility for small molecule separations. Both solvent system development approaches were applied to Heptane-Methyl isobutyl ketone (MIBK)-Acetone-Water, Heptane-Isopropyl acetate (iPAc)-Acetone-Water, HTAW, and HEMWat systems. The separation performance and selectivity of the solvent systems was tested using a model mixture (6). Figure 1 shows how the selectivity for the seven components in the mixture is very similar for the HTAW and HEMWat phase systems.

Figure 1: LogKD of the 7 components in the mixture plotted against the water content of the lower phase of the HTAW and HEMWat phase systems. Acknowledgements The TSB-STEP team would like to acknowledge financial support from the Technology Strategy Board’s High Value Manufacturing programme (Grant No. TSB-HVM/BD506K). References

1. Berthod, A.; Hassoun, M.; Ruiz-Angel, M.J. Anal. Bioanal. Chem. 2005, 383, 327-340. 2. Garrard, I.J. J Liq. Chrom. & Rel. Technol. 2005, 28, 1923-1935. 3. Wu, D.; Jiang, X.; Wu, S. J. Sep. Sci. 2012, 33, 67-73. 4. Garrard, I.J.; Janaway, L.; Fisher, D. J. Liq. Chrom. & Rel. Technol. 2007, 20, 151-163. 5. Renault, J.H.; Nuzillard, J.M.; Intes, O.; Maciuk, A. In: Countercurrent chromatography, the support-free liquid stationary phase (Comprehensive analytical chemistry, vol. 38) Berthod A (ed) Elsevier, Amsterdam, 2002; pp 49-83. 6. Ignatova, S.; Sumner, N.; Colclough, N.; Sutherland, I. J. Chrom. A. 2011, 1218, 6053-6060. 7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-30 Large-Scale Preparative Profiling of Anacardic Acids in Cashew Nut Shell Oil Liquid by Spiral-Coil Countercurrent Chromatography and Off-Line ESI-MS-MS Continuous Infusion Mariana Neves Vieira 1 , Kathrin Welke 1 , Josué A. Murillo-Velásquez 2 , Suzana Guimãres Leitão 3 , Peter Winterhalter *,1 , Gerold Jerz 1 1 Institute of Food Chemistry, Technische Universität Braunschweig, Braunschweig, Germany, fax: +49-531-391-7230, * [email protected] 2 Universidad De El Salvador, Escuela de Química, El Salvador 3 Faculty of Pharmacy, Federal Univeristy of Rio de Janeiro, Brasil Keywords: spiral-coil CCC, off-line ESI-MS/MS coupling, Anacardium occidentale oil

Anacardium occidentale (Anacardiaceae) is known as an ethnomedicinal remedy. Beside its edible fruits, the cashew nuts are high value products for Latin America. Since the 1950s, residual cashew nut shell oil liquids (CNSL) were used as a source for anacardic acid (aa) recovery in industrial processes. Various interesting biological activities had been found by in-vitro assays. Anti-tumor (1), and antibacterial activities of ‘aa’ against methicillin-resistant Staphylococcus aureus (2,3), and Helicobacter pylori should be mentioned (4). Also activity against Malaria (Plasmodium falciparum) (5), and ‘tropical neglected diseases’, such as Chagas Disease (Trypanosoma cruzi) (6), and Leishmaniosis (Leishmania sp.) (7) were described.

Heat-processed CNSL was separated by a large-volume spiral coil countercurrent chromatography (CCC) prototype

(volume: 5.5 L, flow rate: 15 mL/ min, injection: 30 g CNSL). Operated with the biphasic solvent system n-hexane/ acetonitrile [2:1 (v/v)], the spiral-coil CCC fractions were off-line infused to an ESI-MS-MS device (Bruker HCT ion trap-MS). This method yielded a reconstituted metabolite profile and finally enabled a MS-target-guided isolation procedure (Figure 1). Prior to the off-line infusion the fractions were diluted by the factor 50. Elution orders and co-elution effects of minor and major concentrated anacardic acids were specifically monitored by selected ion-traces (neg. mode, scan: m/z 50-1500, [M-H] - signals) (8). The use of the spiral coil-CCC enabled the specific recovery of homologue anacardic acids in the gram range and will be of great value for biological evaluations. Minor constituents were significantly enriched for later 1D/2D-NMR spectroscopic analysis.

C O O H H O R

C15 anacardic acids C17 anacardic acids R= : R= : 11 17 1 : m /z 341 5 : m /z 369 8 14 10 11 15 17 2 : m /z 343 6 : m /z 371 8 10 15 3 : m /z 345 17 7 : m /z 373 8 15 10 4 : m /z 347

Figure 1.ESI-MS selected anacardic acid ion traces from the continuous off-line infusion of spiral CCC-fractions (1 min in the reconstituted ESI-MS trace is equivalent to a CCC-elution volume of 180 mL)

References:

1. Kubo et. Al., J. Agric. Food Chem. 1993, 41, 1012-1015. 2. Muroi et al., Biosci. Biotech. Biochem. 1994, 58, 1925-1926. 3. Kubo et al. J. Agric. Food Chem. 2003, 51, 7624-7628. 4. Castillo-Juárez et al., J. of Ethnopharmacol. 2007, 114, 72–77. 5. Lee et al., Parasitol. Res. 2009, 104, 463–466. 6. Pereira et al., Bioorg. Med. Chem. 2008, 16, 8889-8895. 7. Franca et al, Rev. Soc. Bras. Med. Trop. 1996, 29, 229-232. 8. Jerz et al., In: Recent Advances in the Analysis of Food and Flavors, pp 145-165. ACS Symposium Series 1098; American Chemical Society: Washington, DC, 2012.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-31

Preparative Metabolite Profiling of the Diatom Microalga Phaeodactylum tricornutum by High-Speed Countercurrent Chromatography and Continuous APCI-MS-MS Infusion

Beate Bonsch 1, Robert Dillschneider 2, Rosa Rosello 2, Clemens Posten 2, Peter Winterhalter *, 1, Gerold Jerz 1 1 Institute of Food Chemistry, Technische Universität Braunschweig, Braunschweig, Germany, fax: +49-531-391-7230, * [email protected] 2 Institute of Process Engineering in Life Sciences, Department of Bioprocess Engineering, Karlsruhe Institute of Technolgy (KIT), Karlsruhe,Germany Keywords: Phaeodactylum tricornutum, diatom microalga, preparative HSCCC-off-line-APCI-MS-MS

Phaeodactylum tricornutum is a microalga (diatom) known to produce free fatty acids (FFAs) with anti-bacterial properties. FFAs, such as (6Z,9Z,12Z)-hexadecatrienoic acid, eicosa-pentaenoic acid (EPA), hexadecatrienoic acid (HTA), and palmitoleic acid (PA) had been proven for their strong anti-bacterial activities, e.g. against methicillin-resistant Staphylococcus aureus (MRSA) (1-3). Especially, in areas where conventional antibiotics are prohibited, such as in agriculture, and food preservation, or extensive use of antibiotics should be prevented, the anti-bacterial FFAs could be highly valuable and safe additives. Biofilm formation caused by bacterial adhesion in medicinal products with resulting severe nosokomial hospital infections could possibly be reduced by surface coatings with FFAs. (1-3). Since almost 20 years, high-speed countercurrent chromatography (HSCCC) had been proven to be an excellent method for purification of natural products on larger lab-scale. P. tricornutum was cultivated in a 12L bubble photobioreactor, and the biomass was freeze-dried. The lipophilic extract was injected to a preparative triple-coil HSCCC (PTR 1000, volume: 850 mL, mobile phase flow rate: 3.0 mL/ min, injection: 800 mg). The separation mode was ‘head-to-tail’ with the biphasic solvent system n-hexane/ acetonitrile 1:1 (v/v), and the elution- and extrusion-approach (two-column-volume method) of Berthod et al. resulted in the fractionation of the complete polarity profile of the injected sample (4). For a more specific and mass-spectrometric target-guided isolation of bioactive metabolites, HSCCC separated fractions were monitored by a fast off-line infusion method to APCI-MS-MS. This delivered important structural data also as basis for exact compound fractionation (5). The off-line infusion monitored the 6 hrs elution-extrusion HSCCC-run in a 50 min single APCI-MS-MS data file. This approach largely reduced the APCI-MS-operation time in comparison to a direct on-line-HSCCC-MS coupling experiment (5). The APCI-MS-MS system was set to monitor the most abundant 10 precursor ions with its respective MS/MS fragment ions. Partly, strong compound co-elution effects were perceived, but elucidated the identity of the carotenoid fucoxanthin (1) (6), free fatty acids (2), hydroxy-phaeophytine (3), diglycerides (4), and triglycerides (5) in P. tricornutum (cf. Figure 1).

Figure 1. Ion-traces of preparative HSCCC by off-line APCI-MS-MS (pos mode) infusion: m/z [M+H]+

References

1. Desbois et al., JMBA 2010, 90(4), 769–774, 2. Desbois et al., Appl. Microbiol. Biotechnol. 2008, 81:755–764. 3. Desbois and Smith, Appl. Microbiol. Biotechnol. 2010, 85:1629–1642. 4. Berthod et al., Anal. Chem. 2003, 75: 5886-5894. 5. Jerz et al., In: Recent Advances in the Analysis of Food and Flavors, pp 145-165. ACS Symposium Series 1098; American Chemical Society: Washington, DC, 2012. 6. Kim et al., Appl. Biochem. Biotechnol. 2012, 166:1843–1855.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-32 Zebrafish bioassay-guided isolation of anti-angiogenic components by high-speed counter-current chromatography from licorice

Liwen Han, Yanqiang Yuan, Qiuxia He, Xiqiang Chen, Kechun Liu*

Key Laboratory For Biosensors of Shandong Province, Biology Institute of Shandong Academy of Sciences, Jinan, 250014, China E-mail: [email protected]

Keywords: Zebrafish/ Glycyrrhiza uralensis Fisch./ licorice/ high-speed counter-current chromatography (HSCCC)/ isoliquiritigenin(ISL)/ isolicoflavonol(ISF)

Natural products are one of the most important sources of lead compounds in drug discovery. The advanced isolation technique of lead compounds of natural origin with therapeutical relevant bioassays wound be capable of enhancing work efficiency from complex multi-constituent extracts. In this study, therefore, a bioassay-guided isolation strategy combined with bioactivity screening was explored to identify novel inhibitor from Glycyrrhiza uralensis Fisch(licorice) against aniogenesis, on the basis of zebra fish model and rapid preparative separation of high-speed counter-current chromatography (HSCCC). Zebrafish embryos at 24 hpf (hours post fertilization ) was chosen as the angiogenesis inhibition model for bioactivity screening and evaluation of different extracts and compounds. Firstly, two bioactive fractions, Fr5 and Fr6 were obtained from the ethyl acetate extract from licorice and 14 main compounds were found in Fr5 and Fr6 by HPLC. Then HSCCC method was used for isolation these compounds from Fr5 and Fr6. Using the optimization of separation conditions, n-Hexane-ethyl acetate-methanol-water (5:6:5:5,v/v) system were successfully selected to separate compound 1-5, and n-Hexane-ethyl acetate-methanol-water (6:5:6:5,v/v) was used for the separation of compound 6-10 from Fr5. And using n-Hexane-ethyl acetate-methanol-water (5:5:5:6, v/v) system were successfully selected to separate compound 11-13, and n-Hexane-ethyl acetate-methanol-water (7:5:7:5,v/v) was used for the separation of compound 14. Except a mixture (compound 9+10), 12 single compounds(1-8,11-14) with purity of 90%-98% were obtained in the HSCCC separation process. Since blood circulation and vascular outgrowth in intersegmental vessel (ISV) in zebrafish model were found to be simultaneously inhibited by isoliquiritigenin(ISL) and isolicoflavonol(ISF) in a dose-dependent manner, therefore, these two chemicals were identified and considered as active inhibitors against aniogenesis. These experimental results indicated that zebrafish bioassays combined with HSCCC may provide an alternative pathway for rapid isolation of bioactive natural products.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-33 High-speed counter-current chromatography preparative isolation and purification of four xanthone glycosides from tibetan medicinal plant Halenia elliptica

Li Yulin*a ,Liu Yonglinga,b, Wang Pinga,b, Chen Taoa,b, Zhao Xiaoa, Chen Chena, Sun Jinga

aNorthwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, PR China bGraduate School of the Chinese Academy of Sciences, Beijing 100049, PR China *Corresponding author. E-mail address: [email protected]; Fax: 0971-6143282

Key words: Halenia elliptica, HSCCC, xanthone glycosides

Mainly distributed in Tibet and Qinghai, Halenia elliptica has traditionally been used in Tibetan folk medicine to treat hepatitis [1]. According to prior phytochemical investigations, the principally effective components in Halenia elliptica include triperpenoids, flavonoids, and xanthones[2]. Of these components, xanthone compounds have proven to have broad pharmacological activities. These compounds include: 1-O-primeverosyl-2,3, 4,5,7-pentamethoxyxanthone (C1),1-O-primevero-syl-2,3,4,7-tetramethoxyxanthone (C2), 1-O-prime-verosyl-2,3,5-trimethoxyxanthone (C3), and 1-O-primeverosyl-2,3,4,5-tetramethoxy-xanthone (C4). Conventional methods of preparative separation and purification of these xanthones from Halenia [3,4] elliptica require multiple chromatographic steps on silica gel, C18, and Sephadex LH-20, etc .

In this study, we successfully employed HSCCC to isolate and purify four xanthone glycosides from Halenia elliptica at high purity rates. The experiment obtained the compounds from a 100 mg of crude sample in a one-step separation, and used a two-phase solvent system composed of chloroform–methanol–water (5:3:2, v/v/v). The following represent the compounds obtained: C1 (2.5 mg), C2 (7.0 mg), C3 (10.0 mg), C4 (8.5 mg). For each of the target compounds, the purity was over 95%. The experiment results indicate that HSCCC was an efficient technique for the preparation of pure bioactive compounds from the natural plants.

Fig.1. HSCCC chromatogram of the extract using the solvent system chloroform-methanol-water with the volume ratio of 5:3:2.

Table 1. The K-values of the target compounds measured in several solvent systems Solvent system (V/V/V/V) K（Partition coefficient） C1 C2 C3 C4 ethyl acetate- n-butanol-water (4:1:5) 0.07 0.17 0.38 0.39 ethyl acetate- n-butanol-water (4:2:5) 2.53 1.78 1.01 1.93 ethyl acetate- n-butanol-water (4:4:5) 0.53 0.79 2.34 1.56 chloroform-methanol-water (4:5:4) 0.93 0.47 0.39 0.34 chloroform-methanol-water (5:4:2) 1.03 0.62 0.56 0.48 chloroform-methanol-water (5:3:2) 1.80 0.95 0.64 0.41 References 1. Yang, Y.C., Book of Tibetan Medicine, Qinghai People’s Press, Xining, 1991, p. 111-112. 2 Song, W.Z., General survey on medicinal plants of Gentianaceae in China. Chin J Chin Mater Med 1986, 11(11): 3-7. 3. Rodriguez, S.; Wolfender, J.L.; Odontuya, G.; Purev, O.; Hostettmann, K., Xanthones, Secoiridoids and Flavonoids from Halenia corniculata. Phytochemistry 1995, 40(4): 1265-1272. 4. Sun,H.F.; Hu, B.L.; Ding, J.Y.; Fan,S.F., Three xanthone glucosides from Halenia elliptica. J. Integr. Plant. Biol. 1987, 4: 422-428.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-34 A Combination of Ultrahigh pressure-Assisted Extraction with High-Speed Counter-Current Chromatography for the Preparation of Cantharidin from the blister beetle Xiaojing Lin 1, Hongjing Dong 1,2, Yan-ling Geng 1, Feng Liu 1, Daijie Wang 1, Xiao Wang 1*

1 Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China 2 College of Pharmacy, Shandong University of Traditional Chinese Medicine, University Science and Technology Park in Changqing, Jinan, Shandong 250355, China *Correspondence: Dr. Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, 250014 Jinan, P. R. China. E-mail: [email protected], Fax: +86-531-82964889

Abstract Cantharidin, a well-known natural toxin and vesicant, is extracted from the blister beetle of Mylabris phalerata Pallas or Mylabris cichorii Linnaeus. Cantharidin is the active terpenoid compound of blister beetle and displays many kinds of helpful bioactivities in vivo or in vitro. An efficient method was built successfully for the rapid extraction, separation and purification of cantharidin from the blister beetle by ultrahigh pressure-assisted extraction (UPE) in combination with high-speed counter-current chromatography (HSCCC). The UPE conditions including, extraction time, extraction pressure, solvent ratio were optimized with an orthogonal test. The crude acetone extraction was separated by silica gel column with pure chloroform as elution solution. The separated fraction containing cantharidin was further purified by HSCCC with a two-phase solvent system composed of petroleum ether–acetate–methanol–water

(1.2:0.8:0.9:1.1, v/v). 15.2 mg of cantharidin was obtained from 200 mg of separated fraction with the purity of 99.1% as determined by HPLC. The chemical structure of cantharidin was identified by ESI-MS, 1H-NMR and 13C-NMR.

Keyword: Cantharidin; High-speed counter-current chromatography; Ultrahigh pressure-assisted extraction

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-35 Continuous separation of capsaicin and dihydrocapsaicin using sequential centrifugal partition chromatography Johannes Goll, Andreas Frey, Mirjana Minceva*

Chair of Separation Science and Technology, Friedrich Alexander University Erlangen-Nuremberg, Germany * Egerlandstraße 3, 91058 Erlangen, Fax: 09131-85-27441, E-Mail: [email protected]

Keywords: sequential centrifugal partition chromatography, method development, process design In sequential centrifugal partition chromatography (sCPC) a liquid-liquid biphasic system is used to perform continuous cyclic separations of a feed mixture in two fractions.(1) One sCPC cycle includes two steps; one performed in ascending mode and the other in descending mode. The feed is introduced continuously between two adjacent columns of the sCPC, while the products are collected sequentially at the opposite ends of the columns. During the ascending step the upper phase is used as a mobile phase and carrier (solvent) of the feed components, while during the descending step the lower phase is used for the same purposes. The throughput of an sCPC process is much higher than the throughput of the commonly used batch mode of separation. This makes this technique very suitable for preparative application, especially in the cases of expensive fine chemicals, which are difficult to separate because of their physico-chemical similarity. One example is the separation of capsaicin from dihydrocapsaicin, two bioactive components, which are widely used as food additives and drugs.(2) In this work a method for a continuous separation of capsaicin and dihydrocapsaicin using sCPC is presented. The first step in the method development involves the selection of a suitable biphasic liquid system. This was done by a priori prediction of the partition coefficients of both components in the systems of the Arizona solvent system family using COSMO-RS (“Conductor-like Screening Model for Real Solvents).(3) This approach reveals that Arizona system N, containing equal volume fractions of water, heptane, ethyl acetate and methanol, is the most suitable system. Subsequently, the partition coefficients of capsaicin and dihydrocapsaicin in the selected biphasic system were determined from a pulse injection experiments. The pulse injection elution profiles obtained in ascending and descending mode using different mobile phase flow rates were also used for the calculation of the number of separation stages (theoretical plates). For the sCPC experiments a TMB CPC unit (model Armen TMB-250; Armen Instrument, France) with two columns of 125 ml was used. The sCPC runs were carried out at room temperature, atmospheric pressure and a rotational speed of 1700 rpm, using equal volumes of the upper and lower phase in the sCPC columns. The concentration of capsaicin and dihydrocapsaicin in the feed was 5 g/l, each. The duration of the two steps of the sCPC cycle and the feed and mobile phase flow rates, in each step of the sCPC cycle, were selected using the approach proposed in one of our previous works.(4) In order to predict the influence of the sCPC operating parameter and to optimize them, the separation process was modeled using the cell model. For the simulation of the sCPC operation performances the experimentally derived model parameters (partition coefficients and stage number) were used as input data. Using the selected sCPC unit operating parameters a complete separation of capsaicin and dihydrocapsaicin was achieved. Furthermore, the agreement between the simulated and experimentally achieved sCPC performance was more than satisfactory. References

1. Hopmann E.; Minceva M. J.Chromatogr., A 2012, 1229, 140-147. 2. Peng A.; Ye H.; Li X.; Chen L. J. Sep. Sci. 2009, 32, 2967-2973. 3. Hopmann E.; Frey A.; Minceva M. J.Chromatogr., A 2012, 68-76. 4. Völkl J.; Arlt W.; Minceva M. AIChE J, 2012, in press DOI 10.1002/aic.13812.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-36 Separation of six ginsenosides from panax ginseng using high-performance counter-current chromatography Yi Li1,*, Federico Buonocore2, James Barker 2, Stephen J Barton2, Ian A. Sutherland1 and Svetlana Ignatova1 1 Brunel Institute for Bioengineering, Brunel University, Uxbridge, UK * Tel: +44(0)1895 266911, Fax: +44(0)1895 274608, E-mail: [email protected] 2 School of Pharmacy and Chemistry, Kingston University, UK Keywords: High performance counter-current chromatography, Panax ginseng, gradient method

Ginseng (Panax ginseng C. A. Meyer) has been well known to have a variety of ginsenosides that show diverse biological activities. In the present work, a fast and convenient method for the separation and purification of six ginsenosides from Panax ginseng by high-performance counter-current chromatography was successfully developed. One gradient method in normal phase mode was applied in the first separation step for the isolation of ginsenosides with an EtOAc–BuOH–aqueous 5mM ammonium acetate solvent system. The composition ratio of mobile phase changed from 3.5:0.5:4 to 2.5:1.5:4. Ginsenosides Rd, Rg1, Rb2 and Rb1 (column content) were separated in less than 100 minutes with purities of 92%, 90%, 80% and 90% respectively and with Re/Rc co-eluting. The final retention of stationary phase was 77.6%. Methylene chloride–methanol–isopropanol–aqueous 5mM ammonium acetate (6:2:3:4, v/v) solvent system was used for the isocratic separation of ginsenosides Re and Rc in the second separation step. The purity of ginsenosides Re and Rc was assessed by HPLC–DAD to be over 90%. These ginsenosides structures were characterized by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. Ammonium acetate was used to shorten the separation time and eliminate emulsification all together. The salt can be removed by re-dissolving the sample using acetone.

Figure 1.The fractogram of ginsenosides separation using EtOAc–BuOH–aqueous 5mM ammonium acetate solvent system on DE-Spectrum. Elution mode: Linear gradient, Coil volume: 73 mL

Figure 2.The fractogram of ginsenosides separation Re and Rc with methylene chloride–methanol–isopropanol–aqueous 5mM ammonium acetate (6:2:3:4) on DE-Spectrum. Coil volume: 73 mL

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-37 Isolation and Purification of Pheromones from Ground Beetles by Counter-Current Chromatography Wei Jiang, Bin Wu*

Institute of Marine Biology and Natural Products, Department of Ocean Science and Engineering, Zhejiang University, Hangzhou 310058, People’s Republic of China (Phone: +86-571-882-08540; Fax: +86-571-882-08540; E-mail: [email protected]) Keywords: Pheromones; Ground Beetles; Counter-Current Chromatography

Abstract: Counter-current chromatography (CCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water was applied to the isolation and purification of pheromones from ground beetles. One new and one known sex pheromones were obtained from the crude sample in a one-step separation. Their purities were determined by HPLC. The structures of these two compounds were identified by 1D and 2D NMR and MS means. Furthermore, the absolute configurations were determined by analysis of their CD spectrum.

O O

O O

OH OH

. HO HO 1 2

Figure 1.Sex pheromones from ground beetles

References

1. Y. Ito, R.L. Bowman, Science 169 (1970) 54. 2. S. Wu, C. Sun, K. Wang, Y. Pan, J. Chromatogr. A 1028 (2004) 171. 3. Y. Lu, C. Sun, Y. Wang, Y. Pan, J. Chromatogr. A 1089 (2005) 258

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The Scientific Committee of CCC 2012 will review all abstracts. Abstracts must be submitted before the June 1, 2012 deadline. Abstract contents are the responsibility of the authors. Abstracts exceeding the one-page limit and not in compliance with these template guidelines or missing sections will be returned. Use RTF file format only and submit by e-mail to [email protected]. Note that abstracts sent by fax or mail cannot be considered.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-38

Separation of bioactive compounds from Dark tea using HSCCC target-guided by α-glucosidase inhibitory activity Qiongxian Ye1, Sheng Yin1, Zhimin Zhao1, Depo Yang1, Longping Zhu1, Leslie Brown2, Dongmei Wang1* 1 School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China 2 AECS-QuikPrep Ltd, Bridgend, S. Wales, CF31 4XZ, UK *Dongmei Wang，Tel:(020)39943042, Email: [email protected]

Keywords: dark tea; HSCCC; polyphenol; α-glucosidase inhibitor Recent studies showed that post-fermented tea-dark tea possesses beneficial hyperglycemia activity [1,2]. In this study, 7 fractions were obtained from the 60% ethanolic extract of dark tea by D101 macroporous absorbent column chromatography, and their inhibitory activities against α-glucosidase were assayed. As the results, Fr. 6 showed strongest activity and was chosen to further separation by HSCCC. Solvent systems were examined according to partition coefficients [3] of target compounds (Figure 1, Table 1), and ethyl acetate-n-butanol-methanol-water-acetic acid (4:1:1:4:0.1, v/v/v/v) system was selected. The experimental parameters of HSCCC were shown in Figure 2. Under these conditions, 120 mg Fr.6 sample was introduced into the HSCCC system and 4.8 mg compound 1 and 5.7 mg compound 2 with purities over 95% (Figure 1) were obtained in one step HSCCC separation. The two compounds have been identified by LC-ESI-MS, 1H NMR and 13C NMR. Table 1. The KD of the target compounds in different ratios of volume of solvent system: ethyl acetate-n-butanol-methanol-water-acetic acid (EA-n-bu-M-W-AcA)

a Solvent systems KD (EA-n-bu-M-W-Ac Compound 1 Compound 2 A) 2: 0: 0: 2: 0.05 5.57 0.57 4: 1: 0: 5: 0.05 1.12 1.43 4: 1: 1: 4: 0.1 2.12 3.06 4: 1: 1: 4: 0.2 2.86 3.55

Figure 1. HPLC chromatograms.(a) Fraction 6 from the 60% ethanolic Figure 2. HSCCC separation of the fraction 6 from the 60% extract of dark tea ; (b) HSCCC fraction of peak B in Figure 2; (c) ethanolic extract of dark tea using two phase solvent system HSCCC fraction of peak A in Figure 2. Conditions: column, Ultimate composed of ethyl acetate-n-butanol-methanol-water-acetic acid at AQ-C18, 250 mm*4.6 mm ID, 5 μm; mobile phase, acetonitrile and 4:1:1:4:0.1 v/v; stationary phase: upper organic phase; coil volume, 0.5% acetic acid aqueous at the gradient program (acetonitrile: 0-20 min, 120 mL; rotary speed, 850 rpm; flow rate, 1.5 mL/min; detection, 5-25%; 20-30 min, 25-30%; 30-35 min, 30-90%; 35-45 min, 90%); 280 nm; sample size,120 mg of the sample dissolved in 5 mL upper Referencesflow rate, 1.0 mL/min; detection wavelength, 280 nm. phase and 5 mL lower phase; retention of the stationary phase, 58.6%.

1. Hou, Y.; Shao, W. F.; Xiao, R.; et al. Pu-erh tea aqueous extracts lower atherosclerotic risk factors in a rat hyperlipidemia model. Exp. Gerontol. 2009, 44, 434-439. 2. Kubota, K.; Sumi, S.; Tojo, H.; et al. Improvements of mean body mass index and body weight in preobese and overweight Japanese adults with black Chinese tea (Pu-erh) water extract. Nutr. Res. 2011, 31, 421-428. 3. Walter D, C. Counter-current chromatography: simple process and confusing terminology. J. Chromatogr. A. 2011, 1218, 6015-6023

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-39 Application of high speed countercurrent chromatography in organic synthesis purification R. Gök, G. Jerz, P. Winterhalter * * Technische Universität Braunschweig, Institute of Food Chemistry, Schleinitzstrasse 20, 38106 Braunschweig, Germany (Fax: +49-531-391-7200, email: [email protected]) Keywords: high-speed countercurrent chromatography, HSCCC, organic synthesis, purification

1,1,6-Trimethyl-1,2-dihydronaphthalene (5, TDN), an off-flavor is found in a variety of wines, especially in Riesling wines. The presence of TDN at high concentrations (up to 200 mg/L) found in bottle-aged Riesling wines can cause a “petroleum” or “kerosene” note in the wine (1, 2). 1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene (4, ionene), an educt for the synthesis of TDN (3) and 3,4-didehydro-b-ionone 2, an educt for the synthesis of known TDN precursors (2) are important compounds for the Riesling wine research. The reaction commonly used to synthesize 3,4-didehydro-b-ionone 2 is a bromination with subsequent debromination step, by which a double bond is inserted in the molecule. (2, 4) Ionene 4 is synthesized starting from b-ionone 1 by an iodine-catalyzed cyclization reaction (3) (Figure 1).

O 1. NBS O O 2. N ,N -Diethylaniline A by-products CC l4, ∆ pyridine, ∆ 1 2 1 O

I2, ∆ B by-products CCl4

1 4 5

Figure 1. Synthesis of 3,4-didehydro-b-ionone 2 and 1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene 4 Interestingly, the application of high-speed countercurrent chromatography (HSCCC) for the purification of only a few organic reaction types including catecholamines (5) and heterocycles (6) has been published. The use of this technique for the separation of products from organic synthesis is rare compared to the reported phytochemical application of HSCCC. This work describes the application of HSCCC as an useful, fast and economic alternative for the isolation and purification of 3,4-didehydro-b-ionone 2 from starting material b-ionone 1 (reaction A), and ionene 4 from TDN 5 (reaction B) by means of reversed-phase elution mode (head-to-tail) and the non-aqueous solvent system (n-hexane / acetonitrile; 1:1, v/v). Traditional purification of these reaction products by silica gel column chromatography is unsatisfactory and demanded large solvent amounts, as well as long separation times causing serious degradation of the products. All separations were performed by HSCCC with a multilayer coil planet centrifuge model CCC-1000 (Pharma-Tech. Research Corp.; Baltimore, MD, USA) equipped with three preparative coils connected in series (total volume: 850 mL, flow rate: 3.0 mL/min, injection: 550 mg or 400 mg) (Figure 2). Fractions were monitored by TLC, visualized under UV light (l 254 nm) and the purity of the relevant fractions were screened by GC/MS. Prior to the off-line injection, fractions were diluted by the factor 5.

Figure 2. HSCCC chromatograms of the reaction mixtures from synthesis A and B

References

1. R. F. Simpson, and G. C. Miller, Vitis, 1983, 22, 51–63. 2. P. Winterhalter, Journal of Agricultural and Food Chemistry, 1991, 39, 1825–1829. 3. P. Miginiac, Synthetic Communications, 1990, 20(12), 1853-1856. 4. H. B. Henbest, Journal of the Chemical Society, 1951, 1074-1078. 5. A. Weisz, S.P. Markey, Y. Ito, Journal of Chromatography A, 1986, 383, 132. 6. R.S.F. Silva, G.G. Leitão, T.B. Brum, A.P.G. Lobato, M.d.C.F.R. Pinto, A.V. Pinto, Journal of Chromatography A, 2007, 1151, 197.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-40 Preparative separation of biflavonoids fromGarcinia kola heckel. seeds by spiral high-speed counter-current chromatography

Chris O. Okunjia,c, Peter I. Awachiea, Maurice M. Iwua; Michel Tchimenea, and Yoichiro Ito*

aInternational Center for Ethnomedicine and Drug Development (InterCEDD), Nsukka, Nigeria. cBioseparation Technology Laboratory, Biochemistry and Biophysics Center, NHLBI, National Institutes of Health, 10 Center Drive, Building 10, Room 8N230, Bethesda, MD 20892-1762, USA. [email protected]

Keywords: Garcinai kola, Biflavonoid, Spiral High-Speed Counter-Current Chromatography

The seeds of Garcinia kola Heckel are a highly valued ingredient in African traditional medicine and have been used in many herbal preparations. Recent pharmacological research indicates that G.kola has significant potential including antimicrobial, hepato-protective, bronchodilator effect, anti-inflammatory and antidiabetic activities. G.kola, characteristically, contains high concentrations of biflavonoids, prenylated benzophenones, xanthone and kolaviron. A number of herbal products derived from G. kola seeds has been manufactured and marketed as dietary supplements or phytomedicines1. Thus, there is need for large quantities of the bioactive constituents of G.kola for further in vivo studies, pharmacological testing, chemical optimization or quality control. The key steps in this preparative separation of the biflavonoids are the optimization of solvent combinations and addition of low concentration of trifluoroacetic acid (TFA) to the solvent system. Using an optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:7:4:7:v/v) containing 0.25% aqueous trifluoroacetic acid, five main biflavanones, i.e. GB-I-glucoside (1) GB-1a (2), GB-1 (3), GB-2 (4), kolaflavonone (5), were successively separated from 1.0g of the seed extract in a one-step separation by Spiral HSCCC method2. The purities of the biflavanones range from 95% to 98% as determined by HPLC. The chemical structures of these biflavanones were elucidated by a combination of spectrometric methods and comparison with reference standards. In comparison to earlier HSCCC separation3, the results revealed that the new spiral HSCCC system yielded high partition efficiency with the best stationary phase retention and a short elution time.

References

1. Okunji CO, Ware TA, Hicks RP, Iwu MM, Skanchy DJ., Planta Med. , (2002). 68(5):440-4.

2. Zeng Y, Liu G, Ma Y, Chen X, Ito Y., J Chromatogr A ( 2011) 48:8715-7

3. Okunji C, Komarnytsky S, Fear G, Poulev A, Ribnicky DM, Awachie PI, Ito Y, Raskin I. J Chromatogr A (2007).1151(1-2):45-50.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-41 Separation of anti-tumor constituents from Kalopanax septemlobus (Thunb.) Koidz by silica gel column and high-speed counter-current chromatography Jing Xu, Xueli Cao*, Lu Yin, Chao Cheng, Hong Ren Beijing Key Lab of Plant Resource Research and Development, School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing 100048, China. *E-mail: [email protected]；Fax: 00-86-10-68984898 Keywords: Kalopanax septemlobus (Thunb.) Koidz; Antitumor constituents; A-549 human lung cancer; High-speed countercurrent chromatography (HSCCC)

Kalopanax septemlobus (Thunb.) Koidz, a traditional Chinese medicine was found to display high inhibitory activity against A-549 human lung cancer cells in vitro and against Lewis lung cancer in vivo in our recent researches. In order to investigate potential anti-tumor compounds from K. septemlobus, the ethanol extract of the stem bark of K. septemlobus was fractioned by extraction with petroleum ether, ethyl acetate and n-butanol successively. According to the results of MTT assay, the petroleum ether fraction was subjected to further separation by combination of silica gel column chromatography and high-speed countercurrent chromatography (HSCCC). Firstly, silica gel column chromatography with a stepwise elution of petroleum ether-ethyl acetate (8:1, 6:1, 4:1, 3:1, 2:1,v/v) was employed for pre-separation, according to the analysis by TLC, two fractions A (660mg) and B (895mg) were obtained in 4:1 and 3:1 elution fraction respectively from 10g of petroleum ether extract. Then, 12.2mg of (-)-asarinin (4) were obtained from fraction A by high-speed counter current chromatography (HSCCC) using the solvent system composed of n-hexane-ethyl acetate-methanol-water (3:1:2:1,v/v); And 11.2mg of xanthyletin (1), 12.5mg hinokinin (2) and 350mg of luvangetin (3) were separated by two-step HSCCC with the solvent system composed of petroleum ether-ethyl acetate-methanol-water ( 3:1:2:1 ,v/v) and ( 8:6:7:7 ,v/v) successively from fraction B. The purities of the separated compounds were all over 95% determined by HPLC. The chemical structures were confirmed by 1H-NMR, 13C-NMR and ESI-MS. Further MTT assay proved the anti-tumor activity of luvangetin (3) against A-549 human lung cancer cells in a dose-dependent manner with IC50 values at 4.28 μg/mL. The anti-tumor activities of others are under investigation.

Figure 1. The structures of compounds separarted from Kalopanax septemlobus (Thunb.) Koidz References

1. Ge Bai, Xueli Cao, Hong Zhang, Junfeng Xiang, Hong Ren, Li Tan, Yalin Tang. Direct screening of G-quadruplex ligands from Kalopanax septemlobus (Thunb.) Koidz extract by high performance liquid chromatography. J. Chromatogr. A. 1218 (2011) 6433–6438. 2. Cristiane de Melo Cazal, Vanessa de Cássia Domingues, Jaqueline Raquel Batalhão, Odair Corrêa Bueno, Edson Rodrigues Filho, Maria Fátima G. Fernandes da Silva, Paulo Cezar Vieira, João Batista Fernandes. Isolation of xanthyletin, an inhibitor of ants’ symbiotic fungus, by high-speed counter-current chromatography. J. Chromatogr. A. 1216 (2009) 4307–4312.

This project is financially supported by Beijing Natural Science Foundation.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-42 Effective and Preparative Separation of Bioactive Flavonoids from Gynostemma pentaphyllum Tea Using Elution-Extrusion Counter-Current Chromatography Zhe Chen 1, Yanbin Lu 2, * 1 Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou 310006, China 2 College of Food and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China *fax +86-571-88071024-7587, e-mail: [email protected] Keywords: Counter-current chromatography; Elution-extrusion; Preparative chromatography; Gynostemma pentaphyllum; Flavonoids. Gynostemma pentaphyllum (Thunb.) Makino (Cucurbitaceae, jiao-gu-lan in Chinese), is a kind of herbal tea, widely distributed in China, Japan, Korea and Southeast Asia in warm and humid environments. Its aerial part is also used as folk medicine, to alleviate various diseases and symptoms including hypertension, cough, migraine, insomnia and diabetes mellitus. Recently, much attention has been received in phytochemical and pharmacological studies on this species, because of the unique structures and various biological activities of the relative compounds.

Recently, elution-extrusion counter-current chromatography (EECCC) was successfully developed to extend the hydrophobicity window of the CCC technique [1-5]. The EECCC method takes full advantage of the liquid nature of the stationary phase in CCC and extrudes the most retained solutes out of the column with acceptable peak resolution. The major advantage of EECCC is that the narrow band widths present inside the column are preserved during the extrusion process, resulting in extremely sharp peaks. With these advantages, it is predictable that the EECCC method will be increasingly applied to the fast separation of complex natural and/or biological samples, especially in natural drug discovery programs. Therefore, in the present work, we applied the elution-extrusion protocol for fast separation of G. pentaphyllum tea extract in a customized CCC column (column volume, VC = 180 mL), producing four flavonoids with high purity and milligram level, as a case study of natural drug separation and processing.

Fig. 1 (A) EECCC separation of G. pentaphyllum extracts using a 180 mL CCC column. Biphasic liquid system: n-hexane/ethyl acetate/methanol/water (5/6/5/6 v/v); flow rate: 2.0 mL/min; revolution speed: 600 rpm; stationary phase retention: 60%; detection: 254 nm. (B) Preparative CCC separation of rutin. Biphasic liquid system: ethyl acetate/n-butanol/water (4/1/5 v/v); flow rate: 2.5 mL/min. References

1. Y. Ito, J. Chromatogr. A 2005, 1065, 145-168. 2. Y.Pan and Y. Lu, J. Liq. Chromatogr. Rel. Technol. 2007, 30, 649-679. 3. Y. B. Lu, R. Liu, C. R. Sun and Y. J. Pan, J. Sep. Sci. 2007, 30, 1313-1317. 4. R. Liu, Y. Lu, T. Wu and Y. Pan, Chromatographia 2008, 68, 95-99. 5. Y. Lu, R. Hu, Y. Pan, Anal. Chem. 2010, 82, 3081-3085.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-43 Preparative separation of polyphenols by high-speed counter-current chromatography

Zhenhua Li, Minhui Li*

Baotou Medical College, Baotou 014040, People’s Republic of China (Phone: +86-472-71678950; E-mail: [email protected]) Abstract:

We have applied a high-speed counter current chromatography (CCC) technique to the separation and purification of polyphenols from Vitis quinguangularis Rehd. Polyphenols were separated in a solvent system composed of n-hexane–ethyl acetate–methanol–water.The chemical structures of two complex polyphenols were elaborated by means of electrospray ionization MS and NMR analysis.

Keywords: High-speed counter current chromatography; Polyphenols; Vitis quinguangularis

HO HO

OH OH OH OH O

OH

OH O O HO O HO

OH OH OH

OH

1 2

Figure 1.Polyphenols from Vitis quinguangularis

References

1. W.D. Conway CounterCurrentChromatography; Apparatus, Theory and Applications VCH, New York (1990) 2. Y. Ito, M.A. Weinstein, I. Aoki, R. Harada, E. Kimura, K. Nunogaki 3. Y. Ito, J. Sandlin, W.G. Bowers J. Chromatogr., 244 (1982), p. 247

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The Scientific Committee of CCC 2012 will review all abstracts. Abstracts must be submitted before the June 1, 2012 deadline. Abstract contents are the responsibility of the authors. Abstracts exceeding the one-page limit and not in compliance with these template guidelines or missing sections will be returned. Use RTF file format only and submit by e-mail to [email protected]. Note that abstracts sent by fax or mail cannot be considered. 7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-44 Isolation of the cycloartane glycosides from Sutherlandia frutescens by HSCCC using the spiral tubing support column Korey Brownstein1, George E. Rottinghaus1, Martha Knight2, Yoichiro Ito3, William R. Folk1* 1. University of Missouri, Columbia MO USA 2. CC Biotech, Rockville MD USA 3. National Institutes of Health, Bethesda MD USA *corresponding author: [email protected], Keywords: high-speed countercurrent chromatography, spiral tubing support (STS) column, Sutherlandia frutescens, cycloartane glycosides, sutherlandiosides

Sutherlandia frutescens (L.) R.Br. (Fabaceae) is an endemic plant of southern Africa widely used in traditional medicines for modu-lating immunity, stress and chronic diseases, cancers and symptoms of HIV andother infections. Major secondary (S. frutescens) metabolites include the cycloartane glycosides (sutherlandiosides A-D), flavonols (sutherlandins A-D), pinitol, canavanine, and γ-amino butyric acid. Sutherlandiosides have been purified from the n-BuOH soluble portion of MeOH leaf extracts by chromatography on silica gel and reverse-phase silica gel (1, 2), but the procedure is laborious and capacity-limited. We have used high-speed countercurrent chromatography (HSCCC) (3, 4) to fractionate n-butanol extracts of S. frutescens and have determined conditions by which suther-landiosides can be substantially purified with good yield. A planetary centrifuge (Conway Centrichrom) mounted with an STS rotor (CC Biotech) (5) filled with 1.6 mm ID FEP tubing, pressed in at radials, of 110 mL volume was employed with a [15:1:3:15] solvent system of ethyl acetate, n-butanol, methanol, and water (EBMW). The rotor was filled with upper phase, 0.6-1.3 g sample of extract of S. frutescens dried leaf powder was injected and the lower phase pumped at 1mL/min, with 820 rpm and 4-min fractions collected. Elution mode was L-i-H and stationary phase retention was 55%. After freeze-drying and resuspension in MeOH, fractions were analyzed on TLC plates and the suther-landiosides were visualized with H2SO4 and p-anisaldehyde. Sutherlandiosides B, C, and D were present in

the middle to last fractions (47-77), well separated from other components, including the sutherlandins, that elute earlier. Use of a solvent system without n-butanol (EMW-8:1:7) caused the sutherlandiosides to elute toward the solvent front, and increasing the content of n-butanol (EBMW-19:5:4:22) caused the sutherlandiosides to elute very late. Fractions were quantitatively and qualitatively analyzed by TLC and HPLC-MS.

(Bulk plant material) (Purified Sutherlandioside B) Sutherlandioside B was recovered in yields of ~0.9% dried material, which compares favorably with the content in bulk material (1). The other sutherlandiosides appeared to be recovered in similar yields. The efficiency and scalability of HSCCC for purification of the sutherlandiosides allows for examination of the biological and biochemical activities of these novel secondary metabolites. Supported by NIH/NCCAM/ODS grants 5U19AT003264 and 1P50AT600273. References 1. Fu et al. Journal of Natural Products 2008,71 (10): 1749-1753. 2. Fu et al. Planta Medica 2009,76 (2): 178-181. 3. Ito, Yoichiro. Journal of Chromatography A 2005, 1065 (2): 145-168. 4. Marston, A.; Hostettmann, K. Journal of Chromatography A2006,1112 (1-2): 181-194. 5. Knight, M.; et al. Journal of Chromatography A, 2011, 1218: 4065-4070.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-45 Preparative separation of alkaloids from Aconitum carmichaeli by pH-zone-refining counter-current chromatography Liu Dahui1, Shu Xikai2, Liu Wei2, Wang Xiao2,3*, Yang Bin3, Huang Luqi3 1. Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences,Kunming 650231, China 2. Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China 3. Institute of Chinese Medical Academy of Chinese Medical Science, 16 Dongzhimennei Street, Beijing 100700, China *Correspondence: Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China. E-mail: [email protected] Fax: +86-531-8296-4889

Keywords: pH-zone-refining counter-current chromatography; Aconitum carmichaeli; alkaloids Aconitum carmichaeli is one of the most useful herbal medicines in China, which has been therapeutically used to treat rheumatic pain, paralysis due tostroke, rheumatoid arthritis and some other inflammations (1). The toxic ingredients and pharmacologically active constituents of A. carmichaeli mainly consist of aconitum alkaloids. These alkaloids are of cardiac function, analgesic effect, anti-tumor effect and anti-inflammatory activities (2). In consideration of such various biologically activities of alkaloids, it is necessary to develop an efficient method to separate and purify large quantities of single alkaloid with high purity for further pharmacological research. In this paper, pH-zone-refining counter-current chromatography was successfully applied to the separation of mesaconitine (I), aconitine (II) and hypaconitine (III) from Aconitum Carmichaeli Debx. A 2.3 g quantity of sample was separated using the two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v), 10 mM triethylamine in organic stationary phase and 10 mM hydrochloric acid in aqueous mobile phase. 45 mg of mesaconitine (I), 202 mg of aconitine (II) and 53 mg of hypaconitine (III) were obtained from 2.3 g of crude extract in a separation. The purities of compounds I, II, and III were 91.0%, 95% and 96%, respectively, as determined by HPLC. The chemical structures of these compounds were identified by ESI-MS and 1H-NMR.

Figure 1. PH-zone-refining counter-current chromatogram of preparative separation of alkaloid compounds from Aconitum carmichaeli Debx; Condition: Two-phase solvent system: petroleum ether-ethyl acetate- methanol-water (5:5:1:9, v/v); Stationary phase: the upper organic phase; mobile phase: the lower phase; flow rate: 2.0 ml/min; revolution speed: 850 rpm; detection wavelength: 254 nm; sample size: 2.3 g of sample dissolved in 5 ml of the lower phase without hydrochloric acid and 15 ml of the upper phase of 5:5:1:9 system; retention of the stationary phase: 35 %.

References 1. F. Gao; Shou-An Zhu; Shi-Jun Xiong; Acta Cryst; 2010; E66, P.1342. 2. Van Cao; Xiaofei Chen; Di·Va Liil; Xin Dong; Guo-Qing Zhang; Vi Feng Chai; J PharmAnal; 2011; 1(2), P.125-134.

*Financial supports from the Natural Science Foundation of China (20872083), the Major National S&T Program (2010ZX09401- 302512) and the Key Science and Technology Program of ShandongProvince are gratefully acknowledged.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-46 Preparative separation and purification of hypocrellins from shiraia bambusicola by high-speed counter-current chromatography and preparative high performance liquid chromatography Meixia Xu, Feng Liu, Xiuli Ma, Hengqiang Zhao, Jianhua Liu, Xiao Wang* Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China *Correspondence: Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China. E-mail: [email protected] Fax: +86-531-8296-4889 Keywords: Shiraia bambusicola; hypocrellins; high-speed counter-current chromatography; preparative high performance liquid chromatography;

Shiraia bambusicola P. Hennigs, an ascomycete parasitic on bamboo twigs, has been commonly used as medicinal fungi for treatment of rheumatism and pneusomia in traditional Chinese medicine (1). The fungus is of great medical importance mainly because of its metabolite- hypocrellins which exhibit photodynamic activity towards bacteria and fungi (2). In previously study, perylenequinone pigments hypocrellin A-D and shiraiachrome A-C have been isolated from S. bambusaicola (3,4,5). In consideration of the biological activities of hyprocellins, it is necessary to develop an efficient method to separate and purify large quantities of hyprocellins with high purity for further pharmacological research. In this research, a HSCCC-PHPLC method was successfully developed for the preparative separation and purification of hypocrellins from Shiraia bambusicola. The crude sample was extracted by 95% industrial ethanol with percolating extraction. It was isolate by HSCCC. Then, PHPLC was used to isolate hypocrellin A and hypocrellin C .

A

B

Figure 1 Figure 2

Fig.1 Isolation of three hypocrellins from Shiraia bambusicola by HSCCC. Solvent system: petroleum ether– ethyl acetate–methanol–01.% acetic acid glacial aqueous solution(1:0.6:0.9:0.7, V/V); sample size: 170 mg; flow-rate: 2 ml/min; detection: 254 nm; revolution speed: 850 rpm.Ⅰ: Hypocrellin B; Ⅱ: Hypocrellin A and Hypocrellin C . Fig.2 Purification of hypocrellin A and hypocrellin C from Shiraia bambusicola by PHPLC. Chromatographic column: Shim-pack PREP-ODS (20 mm×250 mm, 15μm); mobile phase: acetonitrile-0.2% glacial acetic acid aqueous solution (75:25, V/V); flow rate: 8 mL/min; detection wavelength: 254 nm; A: Hypocrellin A; B: Hypocrellin C.

From 170 mg of the crude extracts of Shiraia bambusicola, 29.7 mg hypocrellin A, 5.6mg hypocrellin C and 12.7mg hypocrellin B were obtained with the purity of 96.85%, 95.41%, 96.58%, detected by HPLC. The structures of them were identified by 1H-NMR and 13C-NMR. This is the first report that hypocrellins from the Shiraia bambusicola were successfully isolated and purified by the HSCCC-PHPLC method. It showed that HSCCC-PHPLC was an efficient method for the isolation and purification of the hypocrellins from Shiraia bambusicola. And it is of great value on the further development and utilization of Shiraia bambusicola resources. References 1. Liu,B. Chinese medical fungi, 2nd ed., Shanshi People's Press: Shanshi, 1978. 2. Wang HK; Xie JX; Chang JJ; Hwang KM; Liu SY; Lawrence MB; Jing JB; Lee KH. J. Med Chem. 1992, 35, 2721–2727. 3. Kishi T; Tahara S; Taniguchi N; Tsuda M; Tanaka C; Takahashi S. Planta Med. 1991, 57, 376–379. 4. Lizhen Fang; Chen Qing; Hongjun Shao; Yidong Yang; Zejun Dong; Fei Wang; Wei Zhao; Wanqiu Yang; Jikai Liu. J. Antibiot. 2006, 59, 351–354. 5. Houming Wu; Xiafei Lao; Qiwen Wang; Renlong Lu. J. Nat. Prod. 1989, 52, 948-951. *Financial supports from the Natural Science Foundation of China (20872083), the Major National S&T Program (2010ZX09401- 302512) and the Key Science and Technology Program of ShandongProvince are gratefully acknowledged.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-47 Separation, identification and purification of active constituents of Polygonum cuspidatum sieb. et zucc. by HPLC-PDA-ESI-MSn and HSCCC

Fangyuan Gaoa, b, Gao Fang a, b, c, Tingting Zhoua, b, Ji Lia, b, Guorong Fana, b, *

a, * Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road , Shanghai 200433, PR China b Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433 P.R China c Department of Pharmacognosy, School of Pharmacy, Anhui University of Traditional Chinese Medicine Keywords: Polygonum cuspidatum; HPLC-PDA-ESI-MSn; HSCCC

Polygonum cuspidatum Sieb. et Zucc. is a well known traditional Chinese medicine commonly used for treatment of dermatitis and abscess. The major components of P.cuspidatum are anthraquinones, stilbenes, torachrysons and their derivatives which have each specific pharmaceutical activities. An HPLC-PDA-ESI-MSn method was established for the separation and identification of the constituents in the P.cuspidatum. The chromatographic separation was developed on a DiamonsilTM C18 column (4.6 mm×200mm, 5 μm) using 0.1% aqueous acetic acid and acetonitrile as mobile phase adopting gradient elution mode. A total of fourteen bioactive components were identified by HPLC-PDA-ESI-MSn. The typical chromatogram is shown in Figure 1.

Figure 1. HPLC chromatogram of the 75% ethanol Figure 2. The HSCC chromatogram of the 75% ethanol extract extract of Polygonum cuspidatum of Polygonum cuspidatum.

An HSCCC was applied to prepare and purify the hydrophobic compounds from the 75% ethanol extract of P.cuspidatum by ultrasound assisted microwave extraction. The HSCCC separation was performed on an analytical HSCCC system including an Ito coil with the capacity of 30 mL and a temperature control module. The solvent system was composed of petroleum ether -ethy acetate-methanol-water（3:5:9:3 v/v）. During separating in the head to tail mode, the instrument parameter was adjusted to 1800 rpm for rotation, 25 ℃ for temperature, 1 mL/min for flow rate and 254 nm for detection wavelength. The three compounds, including an unknown compound, emodin and physcion were obtained from peak A, peak B, peak C (in Figure 2), respectively. The purity of each compound is over 98% as determined by HPLC. The aim of the research was to prepare and purify the hydrophobic compounds from the P.cuspidatum by HSCCC based on the identification of these fourteen constituents by HPLC-PDA-ESI-MSn. The application of these combination techniques mentioned above can be utilized in the field of the quality control of TCMs. References 1. Dong, J.; Wang, H.; Wan, L.; Hashi, Y.; Chen, S., Identification and determination of major constituents in Polygonum cuspidatum Sieb. et Zucc. by high performance liquid chromatography/electrospray ionization-ion trap-time-of-flight mass spectrometry. Chinese Journal of Chromatography 2009, 27 (4), 425-430. 2. Xia, A.; Zhang, H.; Jia, J.; Chai, Y.; Zhang, G., Determination of muli-index components of quality control of Polygoni Cuspidati Rhizoma et Radix. Chinese Traditional and Herbal Drugs 2011, 42 (9), 1761-1765. 3. Chu, X.; Sun, A.; Liu, R., Preparative isolation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc by high-speed counter-current chromatography. J. Chromatogr. A 2005, 1097 (1-2), 33-39; 4. Chen, L.; Han, Y.; Yang, F.; Zhang, T., High-speed counter-current chromatography separation and purification of resveratrol and piceid from Polygonum cuspidatum. J. Chromatogr. A 2001, 907 (1-2), 343-346.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-48 Purification of a small lectin from a hydroponic culture media using centrifugal partition chromatography Lukasz Grudzien*a, Derek Fishera, Luisa de Moraes Madeirab, Julian Mab, Ian Sutherlanda, Ian Garrarda a Brunel Institute for Bioengineering, Brunel University, London, UK; b St. George’s, University of London, UK * Fax: (+44) 01895 274 608, e-mail: [email protected] Keywords: protein purification, centrifugal partition chromatography (CPC), aqueous two-phase system (ATPS), phase system selection Centrifugal partition chromatography (CPC) with an aqueous two-phase system (ATPS) was used to purify a lectin from other proteins that were co-secreted into a hydroponic medium. Rhizosecretion is often used in the production of recombinant proteins [1]. The process involves expression of a protein in the roots of a plant and its secretion to a hydroponic media. It has considerable advantage over the extraction of recombinant proteins directly from plant tissues as this latter process involves plant harvesting, tissue maceration and subsequent protein purification. Not only is this process expensive and time consuming, but it can cause the release of many plant contaminants including proteolytic enzymes. Although rhizosecretion eliminates these particular drawbacks, it does creates its own challenges: the yield of proteins obtained from rhizosecretion can be as low as 10μg/ml of the hydroponic media and therefore proteins produced using this method need to be concentrated from a large volume of a hydroponic media.As part of studies to develop a method to extract a lectin protein from a large quantity of aqueous hydroponic media, we were examining ultrafiltration followed by CPC. Ultrafiltration allowed over 100-fold concentration of the protein and elimination of peptides and small molecules. The concentrated material was then processed on CPC to purify the target protein from impurities which were mainly other proteins. Due to its gentle nature, ATPS was selected as the phase system. Therefore, a range of two phase systems was examined to find one which would provide differential partitioning of the target protein and impurities. The effects of seven parameters were studied on partitioning of the protein (determined by ELISA) and impurities (determined by SDS-PAGE). These included: concentration of the polymer and salt (12% and 18%), polymer type (polyethylene glycol (PEG) and poly(ethylene glycol-run-propylene glycol) monobutyl ether), molecular weight of polymer (950 and 4000Da), salt type (sodium citrate sodium sulphate, sodium phosphate, potassium phosphate and ammonium sulphate), pH of the system (3.0 and 7.0), concentration of sodium chloride (0 and 5%) and concentration of sodium perchlorate (0 and 5%). Design-of-Experiment software allowed a reduction of the number of required experiments from 320, if phase systems in all these combinations were tested, to 46. The software was then used to generate two ATPSs which provided differential partitioning of the target protein and impurities. Both ATPSs were used as the phase system in CPC as shown in Figure1; A) ATPS 13/13% PEG4000/potassium phosphate, pH 3 and B) ATPS 13/13% PEG4000/ammonium sulphate, pH 7.

Figure 1. Purification of a lectin protein on CPC. Fr- fractions of the mobile phase, C- coil pump out. Bars represent lectin concentration measured by ELISA. SDS-PAGE in the background shows the protein contaminants.

References [1] Drake, P. et al, (2009) FASEB J, 23; 3581-3589

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-49 Purification of lutein from kale by high speed countercurrent chromatography Pingxiao Duan1, Qingqin Xu2, Lei Zhang1, Xiangdong Wang*1

*1 Food Science and Engineering,College of Engineering,Shan Xi Normal University. [email protected] 1 Food Science and Engineering, College of Engineering, Shan Xi Normal University 2 Analytical Chemistry, Analysis and Testing Centre, Shan Xi Normal University

Keywords: Kale, lutein, purification, High-speed Counter-current Chromatography

Kale is a form of cabbage (Brassica oleracea Acephala Group) which is rich in lutein. Lutein may serve to protect the retina from the ionizing effect of blue light since it is accumulated in human retina. As a food additive, lutein has the E number E161b (INS number 161b) and is approved for use in the EU, Australia and New Zealand. Extraction and purification of lutein from kale can be used as food additive. In the present study, high-speed counter-current chromatography was used for purification of lutein from kale extract.

Fresh kale was smashed and dried for extration of lutein by supercritical carbon dioxide. The extract was subjected to purification of lutein by high-speed counter-current chromatography (HSCCC). Using n-hexane-ethanol-water (6:5:1.5) as a solvent system, one-step HSCCC purification resulted in lutein fraction with a purity of 35.9 % from an initial purity of 24.9% in the extract.

2000

1500

1000

500

0

-500 0 2 4 6 8 10 12 14 16

Fig. 1 HPLC chromatogram of lutein fraction from HSCCC separation

References

[1] Aleman TS, Duncan JL, Bieber ML, et al. (July 2001). "Macular pigment and lutein supplementation in retinitis pigmentosa and Usher syndrome". Invest. Ophthalmol. Vis. Sci. 42 (8): 1873–81.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-50 NewHyphenated CPC-HPLC-DAD-MS strategy for simultaneous isolation, analysis and identification of phytochemicals. Thomas Michel1, Emilie Destandau1, Laëtitia Fougère1, Gregoire Audo*2, Claire Elfakir1

1Institute of Organic and Analytical Chemistry, Université d’Orléans–CNRS, UMR CNRS 7311, BP 67059, 45067 Orléans Cedex 2, France 2Armen instrument, 16 rue Ampère, 56890 Saint Avé, France. [email protected]

Keywords: CPC-HPLC-DAD-MS, xanthones, automation

We present here the development of a versatile tool for fast screening and rapid detection of bioactive natural products from plant extracts: the on-line coupling of centrifugal partition chromatography (CPC)-UV to

HPLC-UV-MS, via a six position switching valve (1). This strategy offers the possibility to get instantly HPLC fingerprint of fractions and structural information about separated molecules during the CPC fractionation step.

This new approach was applied to the fractionation and purification of xanthones from Garcinia mangostana

(Clusiaceae) pericarp. CPC was conducted using the biphasic solvent system heptane/ethyl acetate/methanol/water (2:1:2:1, v/v/v/v) and HPLC separation was done with a monolithic column under reversed phase conditions. The combined CPC-HPLC-DAD-MS allows the simultaneous fractionation, detection and characterization of sixteen molecules. Ten molecules of them were identified based on their UV and MS spectra. Furthermore, the methodology has led to isolation of α-mangostin and γ-mangostin at respectively 98% and 98.5% purity in a very short time.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-51 Impurities preparation and identification of sodium tanshinone II a sulfonate by HSCCC and LC-MSn Chen Shanqiaoa,b, Zhou Tingtinga,*, Liu Chengchu b, Fan Guoronga,* a Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University b College of Food Science and Technology, Shanghai Ocean University *325 Guohe Road, Shanghai 200433, China; fax: +86 21 2507 0388; email: [email protected], [email protected] Keywords: Sodium tanshinone II A sulfonate, Impurity, HSCCC, LC-MSn Sodium Tanshinone II A Sulfonate (STS) is the derivative of Tanshinnone II A, the effective component of traditional Chinese medicine Danshen (the root of Salvia miltiorrhiza Bunge)1. Even though this drug of 30 years’ history has been investigated a lot about synthesis2, the quality control especially quantitative analysis of the impurities was rarely researched 3due to the lack of reference substances. The present work applied single step HSCCC separation in the preparation of 2 impurities which are disproportionately low in STS bulk drug taking the advantages of high sample load, high efficiency and free of inreversible adsorption. Moreover, another impurity was prepared by preparative HPLC after HSCCC enrichment. 6 more impurities which are too low for direct LC- MS detection were identified due to the HSCCC enrichment. The HSCCC separation was performed on a TBE- 300 A semi- preparative system include an Ito coil with the capacity of 300 mL and a temperature control module. The solvent system was composed of chloroform-n butanol-methanol-water (3:0.2:3:2). Additionally 1%(V/V) saturated Ammonium acetate aqueous solution was added as an demulsifier. During separating in the head to tail mode the instrument parameter was adjusted to 800 rpm for rotation, 20 ℃ for temperature, 2 mL/min for flow rate and 280 nm for detect wave length. The fractions were collected every 2 minutes after the solvent front came out by an auto collector. The HSCCC chromatography is shown in figure 1. The 3 shaded groups of fractions were pooled together exclusively while others were recovered respectively. The 3 groups and all the singly recovered fractions were analysed by LC- MSn. Sodium Przewaquinone A Sulfonate (5) in purity of 96.7% and Sodium Hydroxyl Tanshinone Sulfonate (2) with 95.3% were found in group II and III. Sodium 1, 2-dehydrotanshinone II A Sulfonate (8) in 100% purity was obtained by preparative separating of group I. 6 impurities and STS (1) were detected and identified in the other fractions. The conjectured chemical structure are shown in figure 2. This research established a efficient and solvent saving large scale preparation of extremely low impurities from the bulk drug of STS. The enrichment by HSCCC is also be utilized in HPLC preparation and LC-MSn identification. The application of HSCCC is extended to the field of pharmaceutical quality control.

Figure 1. The HSCCC chromatography that the 3 groups of fractions was pooled together Figure 2. The conjectured structure of STS and the impurities respectively

References

1. Sun, A.; Zhang, Y.; Li, A.; Meng, Z.; Liu, R. Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography. Journal of Chromatography B 2011, 879, 1899-1904. 2. CHIEN, M.-K.; YOUNG, P.-T.; KU, W.-H.; CHEN, Z.-X.; CHEN, H.-T.; YEH, H.-C. Studies on the Sctive Principles of Dan-Shen--Ⅰ.the Sructure of Sodium Tanshinone Ⅱ-A Sulfonate and Methylene Tanshinquinone. Acta Chimica Sinica 1978, 199-206. 3. Zou, Q.; Huang, B.; He, T.; Wei, P.; Zhang, Z.; Ouyang, P. Identification, Isolation, and Characterization of Impurities in Sodium Tanshinone IIA Sulfonate. Journal of Liquid Chromatography & Related Technologies 2009, 32, 2346-2360.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-52 Method optimisation for isolating purified α-mangostin from Garcinia mangostana L. rinds using HPCCC

Sri Murhandini1,2*, Emily Keaveney3, Stephen J Barton3, James Barker3, Ian Garrard2, Derek Fisher2 and Svetlana Ignatova2

1. The National Agency of Drug and Food Control of the Republic of Indonesia 2. Advanced Bioprocessing Centre, Brunel Institute for Bioengineering, Brunel University London, UK 3. School of Pharmacy and Chemistry, Kingston University, UK *Correspondence should be addressed to fax: + 441895274608 and e-mail: [email protected] Keywords: α-mangostin, production, multiple injections, HPCCC

Separation and purification techniques using liquid flow processing by Counter Current Chromatography (CCC) are widely applied particularly for the separation of components from plant extracts. However, generally only a single injection of the sample into a CCC apparatus is used due to sample complexity. Multiple injections to increase the overall yield and scale of the purification are seldom used. This has the advantage of reducing solvent consumption and run turnover time, both of which reduce the cost of the purification process. We report a method for the production of α-mangostin from Garcinia mangostana L. rinds with high purity and yield as reference material for quality control and standardisation of mangosteen based products. Separations were conducted at an analytical scale on a Mini High Performance Counter Current Chromatography (HPCCC) instrument with column volume 17.7 mL; using a hexane/ethyl acetate/methanol/water (5:5:10:4 v/v) solvent system in reverse phase at 2100 rpm; 25ºC; and flow rate of 1 mL/min. The extract was prepared by overnight maceration of mangosteen rinds powder in 80% aqueous ethanol at 30°C, with a yield of 10.6% (w/w) of crude mangosteen extract from the rinds. The extract was injected up to 10 times with 25 min interval time between injections without any replacement or topping up the stationary phase. There was little loss of stationary phase after first injection. α-Mangostin was well separated after 10 injections of 9.1 mg extract in 0.86 mL lower phase (~100 mg/mL crude powder) and 5 injections of 22.8 mg extract in 0.86 mL lower phase (~250 mg/mL crude powder), although the resolution of the peaks of the α-mangostin and other target compounds reduced with each injection. The α-mangostin was produced with average 98% purity and 96% yield for the 10 injections and 99% purity and 93% yield for the 5 injections based on quantitative HPLC analysis. Characterisation of the 4 peaks obtained in HPCCC by LC-MS and NMR was also attempted. This optimised method for production of α-mangostin gave high purity and yield with reduced processing time and less solvent use. Although this approach may have general applicability to other extracts, variations in the stability of the selected phases system to the extracts can be expected and this may limit the number and concentration of the samples injected.

160,000,000 220,000,000

200,000,000 140,000,000 α-Mangostin Average 99% purity and 93% yield α-Mangostin Average 98% purity and 96% 180,000,000 120,000,000 160,000,000

100,000,000 140,000,000

α-Mangostin α-Mangostin 120,000,000 80,000,000 Target 2 Target 2 100,000,000 Target 3 Target 3 60,000,000 Target 4 80,000,000 Target 4

60,000,000 40,000,000 Peak Area (uVolt*sec) Area Peak Peak Area (uVolt*sec) Area Peak Peak Area (uVolt*sec) Area (uVolt*sec) Area Peak Peak Peak Area (uVolt*sec) Area Peak Peak Area (uVolt*sec) Area Peak Peak Area (uVolt*sec) (uVolt*sec) Area Area Peak Peak 40,000,000 20,000,000 20,000,000

0 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (min) Time (min)

Figure 1. HPCCC Fractogram vs. Time; 10 injections of Figure 2. HPCCC Fractogram vs. Time; 5 injections of 9.1 mg extract in 0.86 mL LP 22.8 mg extract in 0.86 mL LP

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-53 Preparative isolation and purification of biflavonoids from Ephedra sinica by high-speed counter-current chromatography Jialian Li, Lei Fang, Daijie Wang, Xikai Shu, YangLing Geng, Xiao Wang* Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China *Correspondence: Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China. E-mail: [email protected] Fax: +86-531-8296-4889 Keywords: Ephedra sinica; Biflavones; High-speed counter-current chromatography; Ephedra sinica, a shrub-like plant known as Ma Huang or Ephedra, is a traditional Chinese medicinal herb that has been used for thousands of years (1). Ephedra is generally not used alone, but rather as part of herbal formulas, and is known to be used for treatment to autoimmune and inflammatory diseases with safe and efficient function (2). The original isolated and major active medicinal ingredients of Ephedra stems are alkaloids. Stems contain 1~3% of the total alkaloids found in Ephedra plants, with ephedrine accounting for 30–90% of this total depending on the species. In addition, other active constituents, such as biflavonoids are also found in the stem, which also showed potent bioactivies (3-5). In order to find more bioactive biflavonoids for further pharmacological research, it is necessary to develop an efficient method to separate and purify large quantities of single biflavonoids with high purity. In this research, high-speed counter-current chromatography was successfully applied to the preparative separation and purification of biflavonoids from the stems of E. sinica. The experiment was performed with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.5:3.5:1:3, v/v). 250 mg of the crude extract was purified in one-step separation, yielding 18 mg of mahuannin D, 21 mg of mahuannin A, 32 mg of dihydroquercetin, and 29 mg of (+)-catechin with the purities of 98.1%, 98.5%, 98.7% and 98.8%, respectively, as determined by high-performance liquid chromatography. The structures of the oxindole alkaloids were identified by UV, ESI-MS, 1H NMR and 13C NMR. This is the first report that biflavonoids from the stems of E. sinica were successfully isolated and purified by the high-speed counter-current chromatography method, which demonstrated that HSCCC is a fast, effective and powerful technique for the isolation and purification of biflavonoids from natural herbs.

Figure 1. Separation of biflavonoids from the stems of E. sinica by high-speed counter-current chromatography. Solvent system: petroleum ether–ethyl acetate–methanol–water (0.5:3.5:1:3); sample size: 1.5 g; flow-rate: 1.5 ml/min; detection: 254 nm; revolution speed: 800 rpm. Ⅰ: mahuannin D; Ⅱ: mahuannin A; Ⅲ: dihydroquercetin; Ⅳ: (+)-catechin.

1. Abourashed, E. A.; Eialfy, A. T.; Khan, I. A.; Walker, L. Phytother Res 2003, 17, 703–712. 2. Ray, S.; Phadke, S.; Patel, C., Hackman, R. M.; Stohs, S. Arch. Toxicol. 2005, 79, 330–340. 3. Andraws, R.; Chawla, P.; Brown, D. L. Prog Cardiovascular Dis 2005, 47, 217–225. 4. Friedrich, H.; Wiedemeyer, H. Planta Medica 1976, 30, 223–231. 5. Cottiglia, F.; Bonsignore, L.; Casu, L.; Deidda, D.; Pompei, R.; Casu, M.; Floris, C. Nat. Prod. Res. 2005, 19, 117–123.

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012 P-54 Isolation of Chemical Constituents from Ilex rotunda by High-Speed Counter-Current Chromatography

Chun Wang a, Zhimao Chao a, *, Wen Sun a, Xiaoyi Wu a, Yoichiro Ito b a Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China b Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8014, USA

ABSTRACT Jiubiying, the dried barks of Ilex rotunda Thunb. (Aquifoliaceae) has been used as herbal tea and traditional

Chinese drug for the functions of heat-clearing, detoxifying, dehumidification, and odynolysis. Pedunculoside and syringin are the two main bioactive components. In order to obtain more components for the new drug development, we try to isolate and purify some chemical constituents from Jiubiying by high-speed counter-current chromatography (HSCCC). The two-phase solvent system was composed of ethyl acetate-n-butanol-water at an optimized volume ratio of 1:6:7 (v/v/v). 19.6 Mg dunculoside with a purity of

92.4%, 10.6 mg syringin with a purity of 94.1%, and 3 other compounds were obtained from 300 mg ethanol extract by one run of TBE-300A HSCCC machine (Shanghai Tauto Biotech, Shanghai, China). Their structures were identified on the basis of MS, IR, UV, and extensive NMR studies.

* Corresponding author at: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. Tel.: +86 10 64014411 2869; fax: +86 10 64013996. E-mail address: [email protected] The authors gratefully acknowledge the financial support by the public welfare research special project in State Administration for Quality Supervision and Inspection and Quarantine (No.201210209).

7th international conference on countercurrent chromatography, Hangzhou, August 6-8, 2012