international conference on countercurrent chromatography, … 2012.pdf · 2012-08-22 · 7 th...

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

010010010010


ProgramProgramProgramProgram

JanuaryJanuaryJanuaryJanuary,,,, AugustAugustAugustAugust 6666,,,, 2012012012012222

8888::::30303030 –––– 9999::::00000000 Registration

9999::::00000000 –––– 9999:1:1:1:10000 Opening CCCCCCCCCCCC 2012201220122012 Chairman:Chairman:Chairman:Chairman: Prof.Prof.Prof.Prof. QizhenQizhenQizhenQizhen DuDuDuDu

9999:1:1:1:10000 –––– 9999:2:2:2:20000 Welcome speech from the director of Zhejiang Gongshang University

SessionSessionSessionSession 1111 –––– CCCCCCCCCCCC KeynotesKeynotesKeynotesKeynotes Chirman:Chirman:Chirman:Chirman: Prof.Prof.Prof.Prof. GuoanGuoanGuoanGuoan LuoLuoLuoLuo

09:20-09:5009:20-09:5009:20-09:5009:20-09:50 Ito, Y. pH-zone-refining countercurrent chromatography :Origin, mechanism, procedure and applications

USA

K-1K-1K-1K-1

09:50-10:2009:50-10:2009:50-10:2009:50-10:20Sutherland, I.*; Hewitson. P.;Janaway, L.; Wood, P;Ignatova, S.

Scalable technology for the extraction ofpharmaceuticals (STEP): Outcomes from a yearcollaborative researchprogramme

UK

K-2K-2K-2K-2

10:20-11:0010:20-11:0010:20-11:0010:20-11:00 TeaTeaTeaTea BreakBreakBreakBreak withwithwithwith PosterPosterPosterPoster &&&& ExhibitionExhibitionExhibitionExhibition sessionsessionsessionsession 1111

11:00-11:3011:00-11:3011:00-11:3011:00-11:30 Berthod, A. Terminology for countercurrent chromatographyFrance

K-3K-3K-3K-3

11:30-12:0011:30-12:0011:30-12:0011:30-12:00 Ignatova, S.*; Sutherland, I.

API recovery from pharmaceutical waste streamsby high performance countercurrentchromatography and intermittent countercurrentextraction

UK

K-4K-4K-4K-4

12:00-13:3012:00-13:3012:00-13:3012:00-13:30 LunchLunchLunchLunch breakbreakbreakbreak

mailto:[email protected]


JanuaryJanuaryJanuaryJanuary,,,, AugustAugustAugustAugust 6666,,,, 2012012012012222

SessionSessionSessionSession 2222 –––– CCCCCCCCCCCC InstrumentationInstrumentationInstrumentationInstrumentation IIII Chirman:Chirman:Chirman:Chirman: Prof.Prof.Prof.Prof. IanIanIanIan SutherlandSutherlandSutherlandSutherland

13:30-14:0013:30-14:0013:30-14:0013:30-14:00

Pro, S.; Burdick, T.; Pro, L.;Friedl, W.; Novak, N.; Qiu,F.; McAlpine, J.B., J. BrentFriesen, J.B.; Pauli, G.F.*

A new generation of countercurrent separationtechnology

USA

O-1O-1O-1O-1

14:00-14:2014:00-14:2014:00-14:2014:00-14:20 Berthod, A.*; Faure, K.;Meucci, J.; Mekaoui, N.

A small volume hydrostatic CCC column forfull and quick solvent selection

France

O-2O-2O-2O-2

14:20-14:4014:20-14:4014:20-14:4014:20-14:40

Du, Q.B.; Jiang, H.; Yin, J.;Xu, Y.; Du, W.; Li, B.; Du,Q.*

Construction of a HSCCC apparatus withcolumn capacity of 12 or 15 liters and itsapplication as flash countercurrentchromatography in quick preparation of(-)-epicatechin

China

O-3O-3O-3O-3


SessionSessionSessionSession 3333 –––– CCCCCCCCCCCC InstrumentationInstrumentationInstrumentationInstrumentation IIIIIIII Chirman:Chirman:Chirman:Chirman: PrPrPrProfofofof.... GuidoGuidoGuidoGuido PauliPauliPauliPauli

15:30-15:5015:30-15:5015:30-15:5015:30-15:50 Kostanyan, A. E. Steady-state and non-steady state operation ofcountercurrent chromatography devices

RRRRussia

O-4O-4O-4O-4

15:50-16:1015:50-16:1015:50-16:1015:50-16:10 G. Audo*; Le Quémeneur, C. True moving bed CPC for continuouspurification of natural substances

France

O-5O-5O-5O-5

16:10-16:3016:10-16:3016:10-16:3016:10-16:30 Goll, J.; Frey, A.; Minceva,M.*

Design of a continuous centrifugal partitionchromatography

Germany

O-6O-6O-6O-6

16:30-16:5016:30-16:5016:30-16:5016:30-16:50 Hewitson, P.*; Sutherland, I.;Wood, P.; Ignatova, S.

Intermittent countercurrent extraction: Scale upand an improved bobbin design

UK

O-7O-7O-7O-7

18:00-19:3018:00-19:3018:00-19:3018:00-19:30 WelcomeWelcomeWelcomeWelcome dinnerdinnerdinnerdinner atatatat aaaa rrrrestaurantestaurantestaurantestaurant





February,February,February,February, AugustAugustAugustAugust 7,7,7,7, 2012201220122012

SessionSessionSessionSession 4444 –––– CCCCCCCCCCCC TheoryTheoryTheoryTheory IIII Chirman:Chirman:Chirman:Chirman: Prof.Prof.Prof.Prof. AlainAlainAlainAlain BerthodBerthodBerthodBerthod

09:00-09:2009:00-09:2009:00-09:2009:00-09:20 Frey, A.*; Minceva, M.Selection of the mobile and stationary phase insupport free liquid-liquid chromatographyapplying a predicative thermodynamic model

Germany

O-8O-8O-8O-8

09:20-09:4009:20-09:4009:20-09:4009:20-09:40Schwienheer, C.*;Adelmann,S.; Merz, J.;Schembecker, G.

Selection and use of aqueous two phase systemsin centrifugal partition chromatography

Germany

O-9O-9O-9O-9

09:40-10:0009:40-10:0009:40-10:0009:40-10:00 Wei, Y.*; Wang, F.; Wang, S. Modeling counter-current chromatography usinga temperature dependence plate model

China

O-10O-10O-10O-10

10:00-10:2010:00-10:2010:00-10:2010:00-10:20 Wu, S. ; Wu, D. ; Liang, J. ;Berthod, A.*

Modeling gradient elution in CCC: Efficientseparation of tanshinones

China,France

O-11O-11O-11O-11


SessionSessionSessionSession 5555 –––– CCCCCCCCCCCC TheoryTheoryTheoryTheory IIIIIIII Chirman:Chirman:Chirman:Chirman: Prof.:Prof.:Prof.:Prof.: PeterPeterPeterPeter WinterhalterWinterhalterWinterhalterWinterhalter

11:00-11:2011:00-11:2011:00-11:2011:00-11:20Merz, J.*; Schwienheer, C.;Adelmann, S.; Schembecker,G.

Investigation of hydrodynamics in CPCchambers using image processing andcomputational fluid dynamics

Germany

O-12O-12O-12O-12

11:20-11:4011:20-11:4011:20-11:4011:20-11:40Chen, X.; Huang, X.; Wang,G.; Zhang J.; Di, D. *

Research of interaction between ionic and teapolyphenols by computer simulation assistedhigh-performance counter-currentchromatography

China

O-13O-13O-13O-13

11:40-12:0011:40-12:0011:40-12:0011:40-12:00Shehzad, O.; Khan, S.; Ha,I.J.; Lee, K.J.; Tosun, A.;Kim, Y.S.*

Modeling of step wise gradients in HSCCC; Arapid and economical strategy for one stepseparation of eight dihydropyranocoumarinsfrom Seseli resinousm.

Korea

O-14O-14O-14O-14

12:00-13:3012:00-13:3012:00-13:3012:00-13:30 LunchLunchLunchLunch breakbreakbreakbreak









SessionSessionSessionSession 6666 –––– CCCCCCCCCCCC ApplicationApplicationApplicationApplication IIII (Room(Room(Room(Room A)A)A)A) Chirman:Chirman:Chirman:Chirman: Prof.Prof.Prof.Prof. XueliXueliXueliXueli CaoCaoCaoCao

13:30-13:5013:30-13:5013:30-13:5013:30-13:50Winterhalter, P.*; Jerz, G.;Kuhnert, S.; Juadjur, A.;Macke,S.

Isolation of bioactive polyphenols fromby-products of food processing usingcountercurrent chromatography

Germany

O-15O-15O-15O-15

13:50-14:1013:50-14:1013:50-14:1013:50-14:10Chen, X.; Huang, X.; Wang,X.; Shi, M.; Wang, G.;Zhang, J.; Di, D.*

Study on preparative separation and purificationof chemical compounds from plants by HSCCC

China

O-16O-16O-16O-16

14:10-14:3014:10-14:3014:10-14:3014:10-14:30 Zheng, Y.; Tong, S. Q.*; Yan,J. Z.

Enantioseparation of 2-phenylpropionic acid byrecycling high-speed countercurrentchromatography using hydroxypropyl-β-cyclodextrin as chiral selector

China

O-17O-17O-17O-17

14:30-14:5014:30-14:5014:30-14:5014:30-14:50 Jin,Y. Y.; Qian, D. Y.; Du, Q.Z.*

Excellent fraction effect of HSCCC on thecompleted separation of amide compounds inblack pepper

China

O-18O-18O-18O-18

SessionSessionSessionSession 7777 –––– CCCCCCCCCCCC ApplicationApplicationApplicationApplication IIIIIIII (Room(Room(Room(Room B)B)B)B) Chirman:Chirman:Chirman:Chirman: PrPrPrProfofofof.... EiichiEiichiEiichiEiichi KitazumeKitazumeKitazumeKitazume

13:30-13:5013:30-13:5013:30-13:5013:30-13:50 Hummel, H. E.*; Hein, D. F.;Ma, Y.; Ito, Y.

Multifunctional tetranor triterpenoids fromAzadirachta indica : A separation challengesolved by MLCCC at both the analytical andpreparative scale

Germany

O-19O-19O-19O-19

13:50-14:1013:50-14:1013:50-14:1013:50-14:10 Hu, R.; Chen, P.; Pan, Y.*

Bioassay guided separation of bioactivecomponents in natural products viacountercurrent chromatography coupled withLC/MS/MS

China

O-20O-20O-20O-20

14:10-14:3014:10-14:3014:10-14:3014:10-14:30

He, Z. C.; Ye, Q. X.; Wu,J.Y.; Yang, D. P.; Brown, L.;Jiang, L.; Zhu, L. P.; Wang,D. M.*

Separation and purification of quanternaryprotoberberine alkaloids from Corydalissaxicola Bunting by HSCCC

China

O-21O-21O-21O-21

14:30-14:5014:30-14:5014:30-14:5014:30-14:50 Yang, M.; Xu, X.*; Xie, Z.;Huang, J.; Xie, C.

Separation and purification of four lignans fromFructus arctii by high-speed countercurrentchromatography

China

O-22O-22O-22O-22







SessionSessionSessionSession 8888 –––– CCCCCCCCCCCC ApplicationApplicationApplicationApplication IIIIIIIIIIII (Room(Room(Room(Room A)A)A)A) Chirman:Chirman:Chirman:Chirman: PrPrPrProfofofof.... HansHansHansHans E.E.E.E. HummelHummelHummelHummel

15:30-15:5015:30-15:5015:30-15:5015:30-15:50 Shibukawa, M.*; Shimizu,K.; Saito, S.

Separation andenrichment of rare earth elementsby stepwise pH gradient countercurrentchromatography with a polyethyleneglycol-Na2SO4 aqueous two-phase system

Japan

O-23O-23O-23O-23

15:50-16:1015:50-16:1015:50-16:1015:50-16:10 Ma, J.; Niu, L.; Cai, X.; Ito,Y.; Yang, F.

Application of high-speed countercurrentchromatography, TLC and LC-MS/MS inlipidomics

China

O-24O-24O-24O-24

16:10-16:3016:10-16:3016:10-16:3016:10-16:30 Xu, M.; Du, Q.;* Shen, L.;Wang, H.

Isolation of two new diterpenoids fromClerodendrum kaichianum hsu. by high-speedcounter-current chromatography using stepwiseelution

China

O-25O-25O-25O-25

16:30-16:5016:30-16:5016:30-16:5016:30-16:50Wang, K. B.; Liu, F.; Liu,Z.L.*, Huang, J. A.; Lin,Y.; Gong, Y. S.

Preparative isolation of Oolong tea polyphenols byhigh-speed countercurrent chromatography

China

O-26O-26O-26O-26

SessionSessionSessionSession 9999 –––– CCCCCCCCCCCC ApplicationApplicationApplicationApplication IV(RoomIV(RoomIV(RoomIV(Room B)B)B)B) Chirman:Chirman:Chirman:Chirman: PrPrPrProfofofof.... ArtakArtakArtakArtak KostanyanKostanyanKostanyanKostanyan

15:30-15:5015:30-15:5015:30-15:5015:30-15:50 Ramos, R. E.; Pauli, G. F.;Chen, S. N.*

Generation of knock-out extracts bycountercurrent chemical subtraction

USA

O-27O-27O-27O-27

15:50-16:1015:50-16:1015:50-16:1015:50-16:10 Wang, W.*; Le, S.; Zhao, X;Wang, T.; Zhang, J.

Rapid separation of polyphenols from green tea byHSCCC and characterization the interactionbetween EGCG and protein by ACE

China

O-28O-28O-28O-28

16:10-16:3016:10-16:3016:10-16:3016:10-16:30 Wei, Y.*; Huang, W.

Online isolation and purification of four phthalidecompounds from Rhizome chuanxiong usingcountercurrent chromatography coupled withsemipreparative high performance liquidchromatography

China

O-29O-29O-29O-29

16:30-16:5016:30-16:5016:30-16:5016:30-16:50He, M.; Du, W.; Du, Q.B.;Zhang, Y.; Li, B.; Ke, C. ;Du, Q.*

Separation of 11-cis-retinal from the retinalisomers by flash countercurrent chromatography

China

O-30O-30O-30O-30

18:00-19:3018:00-19:3018:00-19:3018:00-19:30 GalaGalaGalaGala dinnerdinnerdinnerdinner atatatat aaaa rrrrestaurantestaurantestaurantestaurant


WendesdayWendesdayWendesdayWendesday,,,, AugustAugustAugustAugust 8888,,,, 2012201220122012

SessionSessionSessionSession 10101010 –––– CCCCCCCCCCCC ApplicationApplicationApplicationApplication VVVV (Room(Room(Room(Room A)A)A)A) Chirman:Chirman:Chirman:Chirman: PrPrPrProfofofof.... JohnJohnJohnJohn B.B.B.B. FriesenFriesenFriesenFriesen

09:00-09:2009:00-09:2009:00-09:2009:00-09:20Qiu, F.; Samuel Pro,S. ; Pauli, G. F.; Friesen, J.B.*

NMR-guided countercurrent purification andquantification of green tea catechins

USAO-31O-31O-31O-31

09:20-09:4009:20-09:4009:20-09:4009:20-09:40 Yin, L.; Cao, X. L.*; Xu, J.;Cheng, C.; Pei, H. R.

Separation of bioactive components fromGynura divaricata (L.) DC by high-speedcountercurrent chromatography

ChinaO-32O-32O-32O-32

09:40-10:0009:40-10:0009:40-10:0009:40-10:00 Wang, D.J., Du, J. H.*; Lin,Y. L.; Li, S. B.; Wang, X.

Combination of high-speed countercurrentChromatography and Preparative HPLC toSeparation of Flavonoid Glycosides andCaffeoylquinic Acid Derivatives from Leaves ofLonicera japonica Thunb.


10:00-10:2010:00-10:2010:00-10:2010:00-10:20 Song, G.L.; Wang, J.B.;Wang, Y.B.; Du, Q.Z.*

Preparative separation of conjugated linoleicacids (CLA) from camellia oleifera abel usingpH-zone-refining counter-currentchromatography


SessionSessionSessionSession 11111111 –––– CCCCCCCCCCCC ApplicationApplicationApplicationApplication VIVIVIVI (Room(Room(Room(Room B)B)B)B) Prof.Prof.Prof.Prof. YuanjiangYuanjiangYuanjiangYuanjiang PanPanPanPan

09:00-09:2009:00-09:2009:00-09:2009:00-09:20Hu, P.; Liu, M.; Zhang, M.;*Hua, Y.; Zhang, H.; Wang,Y.; Luo, G.*

From medicinal plant to compound: isolation ofshikimic acid from Illicium verum byhyphenated expanded bed adsorptionchromatography and counter-currentchromatography


09:20-09:4009:20-09:4009:20-09:4009:20-09:40 Lu, Y. B.*; Lin, X. J.; Wang,K.W.

Comprehensive separation of lignans fromZanthoxylum planispinum by countercurrentchromatography combined withhigh-performance liquid chromatography/tandem mass spectrometry


09:40-10:0009:40-10:0009:40-10:0009:40-10:00Wang , X.; Dong, H. J.;Yang, B.; Lin, X. J.; Huang,L. Q.*

Large-scale separation of alkaloids fromcorydalis bungeana turca.by pH-zone-refiningcounter-current chromatography


10:00-10:2010:00-10:2010:00-10:2010:00-10:20 Zhu, Y.; Zhan, Y.; Gui, H.;Wei, Q.; Liu, Y.; Xu, T.*

Separation of the synthetic products ofacetylated rhein by high-speed counter-currentchromatography



11:20-11:4011:20-11:4011:20-11:4011:20-11:40 ClosingClosingClosingClosing remarks,remarks,remarks,remarks, EdwardEdwardEdwardEdward ChouChouChouChou andandandand CraftyCraftyCraftyCrafty ChromatographerChromatographerChromatographerChromatographer AwardAwardAwardAwardceremonyceremonyceremonyceremony Prof.Prof.Prof.Prof. QizhenQizhenQizhenQizhen DuDuDuDu

12:00-13:3012:00-13:3012:00-13:3012:00-13:30 LunchLunchLunchLunch breakbreakbreakbreak13:30-15:0013:30-15:0013:30-15:0013:30-15:00 VisitingVisitingVisitingVisiting TeaTeaTeaTea MuseumMuseumMuseumMuseum ofofofof ChinaChinaChinaChina (Bus(Bus(Bus(Bus service)service)service)service)

15:00-16:3015:00-16:3015:00-16:3015:00-16:30 VisitingVisitingVisitingVisiting SilkSilkSilkSilk MuseumMuseumMuseumMuseum ofofofof ChinaChinaChinaChina (Bus(Bus(Bus(Bus service)service)service)service)

16:30-16:30-16:30-16:30- TourTourTourTour aroundaroundaroundaround WestWestWestWest LakeLakeLakeLake andandandand HangzhouHangzhouHangzhouHangzhou citycitycitycity ((((transpotranspotranspotransporrrrtationtationtationtation selfselfselfself service)service)service)service)


K-K-K-K-1111pH-zone-refiningpH-zone-refiningpH-zone-refiningpH-zone-refining countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography :::: Origin,Origin,Origin,Origin, mechanism,mechanism,mechanism,mechanism,

procedureprocedureprocedureprocedure andandandand applicationsapplicationsapplicationsapplicationsYoichiroYoichiroYoichiroYoichiro ItoItoItoIto

Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and BloodInstitute, National Institutes of Health, Bethesda, MD, USA

*Correspondence address: Fax: 301-402-0013; E-mail: [email protected]

Keywords:Keywords:Keywords: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 anincidental finding that bromoacetyl T3 formed an unusually sharp peak during its purification from a syntheticmixture. The cause of this sharp peak formation was found to be the presence of bromoacetic acid in thesample solution, which formed a sharp trailing border in the column due to its non-linear isotherm. Thebromoacetyl T3 was trapped by this sharp acid border and co-eluted as a sharp peak [1]. ThispH-peak-sharpening method was successfully applied for separation of a small amount of DNP-amino acids byadding three organic acids (as spacer) in the sample solution. When the sample size of each component wasincreased 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 whilepreserving 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 (acidor base) which moves through the column at a rate determined by its partition coefficient in the two-phasesolvent system. In order to generalize the application of the method, a new way to form the sharp borderbetween acid and base in the column was developed as follows [3]: 1) Select the two-phase solvent systemwhich gives partition coefficient value (K) of the analyte being close to one. After equilibration in theseparatory funnel, two phases are separated each in a glass container. 2) Then the retainer (organic acid suchas trifluoroacetic acid for acidic analytes and organic base such as triethylamine for the basic analytes) is addedto the upper organic phase while the eluter (inorganic base such as NH3 for the acidic analytes and inorganicacid such as HCl for the basic analytes) is added to the lower aqueous phase. 3) The column is first entirelyfilled with the upper stationary phase followed by sample injection (without pre-quilibration of the column withthe aqueous phase). Then the lower aqueous mobile phase is eluted through the column. Under thisexperimental condition the eluter in the mobile phase gradually neutralizes the retainer present in the stationaryphase to form a sharp retainer trailing border which moves through the column at a rate largely determined bythe molar ratio between the retainer and the eluter. For example,10 mM retainer and 10 mM eluter will form asharp border which moves through the column at a rate close to that of the compound with K = 1, regardless ofthe composition of the two-phase solvent system. This method has been widely applied to the preparativeseparation 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, andsulfonic 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 inthe 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, itprovides the following several important advantages over the conventional CCC: 1) sample loading capacity isincreased over 10 times; 2) fractions are highly concentrated near its saturation level; 3) yields of pure fractionsare improved by increasing a sample size; 4) charged minute compounds are concentrated and detected at thepeak boundaries; 5) elution peaks are monitored with a pH flow meter for the compounds with nochromophore.

ReferencesReferencesReferencesReferences

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.


K-2K-2K-2K-2ScalableScalableScalableScalable TechnologyTechnologyTechnologyTechnology forforforfor thethethethe ExtractionExtractionExtractionExtraction ofofofof PharmaceuticalsPharmaceuticalsPharmaceuticalsPharmaceuticals (STEP):(STEP):(STEP):(STEP): OutcomesOutcomesOutcomesOutcomes

fromfromfromfrom aaaa 3333 yearyearyearyear CollaborativeCollaborativeCollaborativeCollaborative ResearchResearchResearchResearch ProgrammeProgrammeProgrammeProgramme

IanIanIanIan SutherlandSutherlandSutherlandSutherland1111,,,,*,*,*,*, PeterPeterPeterPeter HewitsonHewitsonHewitsonHewitson1111,,,, LeeLeeLeeLee JanawayJanawayJanawayJanaway2222,,,, PhilipPhilipPhilipPhilip WoodWoodWoodWood2222,,,, NeilNeilNeilNeil EdwardsEdwardsEdwardsEdwards2222,,,,DavidDavidDavidDavid RookeRookeRookeRooke2222,,,, GuyGuyGuyGuy HarrisHarrisHarrisHarris2222,,,, DavidDavidDavidDavid KeayKeayKeayKeay2222,,,, KeithKeithKeithKeith FreebairnFreebairnFreebairnFreebairn3333,,,, DavidDavidDavidDavid JohnsJohnsJohnsJohns3333,,,, NathalieNathalieNathalieNathalie

DouilletDouilletDouilletDouillet3333,,,, ChrisChrisChrisChris ThickittThickittThickittThickitt3333,,,, CliveCliveCliveClive MountainMountainMountainMountain3333,,,, BenBenBenBen MathewsMathewsMathewsMathews4444,,,, RolandRolandRolandRoland BrownBrownBrownBrown4444 andandandand SvetlanaSvetlanaSvetlanaSvetlanaIgnatovaIgnatovaIgnatovaIgnatova1111

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, UK3GlaxoSmithKline, Pharmaceuticals R&D Facility, Stevenage, SG1 2NY, UK

4 Pfizer, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK

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

Professor Sutherland will give a general overview of the industrial scale up of countercurrent chromatographyand, in particular, will be reporting on the outcome of a 3 year £1.5m Technology Strategy Board (TSB) fundedresearch programme first introduced at CCC2010 [1] developing a small footprint, versatile, counter currentchromatography purification technology and methodology which can be operated at a range of scales in bothbatch 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 providingflexible, low capital platform technology driving substantial cost efficiency in both drug development and drugmanufacturing processes.Advances in process automation, method development, continuous processing, robustness/ reliability andthroughput will be presented withscale 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 presentationson specific aspects of the TSB-STEP research prorgramme on new solvent systems for rapid methoddevelopment (Svetlana Ignatova and possibly Guy Harris); continuous processing of waste streams (SvetlanaIgnatova) and Intermittent Counter-current Extraction (ICcE – Peter Hewitson)ReferencesReferencesReferencesReferences

[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., ScalableTechnology for the Extraction of Pharmaceutics (STEP): the transition from academic knowhow to industrialreality, Journal of Chromatography A 2011201120112011, 1218, 6114-6121


K-3K-3K-3K-3TerminologyTerminologyTerminologyTerminology forforforfor CCCCountercurrentountercurrentountercurrentountercurrent CCCChromatographyhromatographyhromatographyhromatography

AlainAlainAlainAlain BerthodBerthodBerthodBerthod

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

Corresponding author e-mail: [email protected]

KeywordsKeywordsKeywordsKeywords: Countercurrent chromatography, countercurrent separation, terminology.

Countercurrent chromatography (CCC) is a liquid chromatography technique that uses a support-freeliquid stationary phase. A biphasic liquid system is used, one phase of the system is selected to be themobile phase and the other is the stationary phase. It is difficult to maintain the volume of a liquidstationary 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-liquidseparation 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 inthe column in such a way that it can interact dynamically with mobile phase. The broad diversity ofcountercurrent separation terminology reflects the innovative spirit of the field as well as the global appealof this technique.

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



K-4K-4K-4K-4APIAPIAPIAPI recoveryrecoveryrecoveryrecovery fromfromfromfrom PharmaceuticalPharmaceuticalPharmaceuticalPharmaceutical WasteWasteWasteWaste StreamsStreamsStreamsStreams

bybybyby HighHighHighHigh PerformancePerformancePerformancePerformance CountercurrentCountercurrentCountercurrentCountercurrent ChromatographyChromatographyChromatographyChromatography andandandand IntermittentIntermittentIntermittentIntermittentCountercurrentCountercurrentCountercurrentCountercurrent ExtractionExtractionExtractionExtraction

SvetlanaSvetlanaSvetlanaSvetlana IgnatovaIgnatovaIgnatovaIgnatova1,1,1,1,*,*,*,*, PeterPeterPeterPeter HewitsonHewitsonHewitsonHewitson1111,,,, PhilipPhilipPhilipPhilip WoodWoodWoodWood2222,,,, NathalieNathalieNathalieNathalie DouilletDouilletDouilletDouillet3333,,,, ChrisChrisChrisChris ThickittThickittThickittThickitt3333,,,,DavidDavidDavidDavid JohnsJohnsJohnsJohns3333,,,, KeithKeithKeithKeith FreebairnFreebairnFreebairnFreebairn3333,,,, RolandRolandRolandRoland BrownBrownBrownBrown4444 andandandand IanIanIanIan SutherlandSutherlandSutherlandSutherland1111


2 Dynamic Extractions Ltd, 890 Plymouth Road, Slough, SL1 4LP, UK3GlaxoSmithKline, Pharmaceuticals R&D Facility, Stevenage, SG1 2NY, UK

4 Pfizer, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK

KeywordsKeywordsKeywordsKeywords: Waste Streams, Liquid-liquid Extraction, Scale-up, Counter-current Chromatography, IntermittentCountercurrent extraction, Continuous Processing

When new projects are scaled up from the research and development stage into manufacturing, recovery ordisposal of waste streams becomes more and more important to minimise cost and reduce both theenvironmental and safety impact. Modern telescopic synthesis techniques and their follow-on purificationprocesses are not always efficient enough to prevent loss of active pharmaceutical ingredients (API) in thewaste stream. There are technologies available by which components of such streams can be recovered, treatedor disposed of. However, most of them are based on selective filtration and aim to recover catalysts or reducetoxicity 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 noadditional processing; 3) recycling materials after such processing if required; 4) disposal with energy recoveryand finally 5) benign disposal. This study is addressing 2 & 3.

The drive towards greener processes, process economics, technology availability, and legislation, all play a partin the selection of the best available technology for waste stream processing. One such technology is HighPerformance Counter Current Chromatography (HPCCC) which is being evaluated as new cost effectivecontinuous platform technology as part of a 3 year Technology Strategy Board High Value Manufacturinginitiative entitled “Scalable Technology for the Extraction of Pharmaceuticals” [1-2]. This is an industry ledcollaborative 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 andwaste.

HPCCC method development for waste steam purification is done at the analytical scale with subsequent scaleup to the preparative 1L instrument. This demonstrates the potential of the technology for the recovery of activepharmaceutical ingredients from complex waste streams in both batch and continuous processing modes. Thescalability of the HPCCC process enables optimization work at the laboratory scale to be transferred directly tothe 18L pilot scale HPCCC. Different scenarios of throughput/purity/yield will be presented demonstrating theflexibility of the technology to purify the target compound at the required trial grade and avoiding expensiveclean up processes. This process is therefore capable of reducing the environmental impact of pharmaceuticalwaste streams.

ReferencesReferencesReferencesReferences1. Technology Strategy Board (TSB) High Value Manufacturing Award (Grant No. TP14/HVM/6/I/BD506K) “Scalable Technology for theExtraction of Pharmaceuticals (STEP) September 2009 to August 20122. 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 fromacademic knowhow to industrial reality, Journal of Chromatography A 2011201120112011, 1218, 6114-6121


OOOO-1-1-1-1AAAA newnewnewnew generationgenerationgenerationgeneration ofofofof CCCCountercurrentountercurrentountercurrentountercurrent SSSSeparationeparationeparationeparation TTTTechnologyechnologyechnologyechnology

SamuelSamuelSamuelSamuel ProProProPro1111,,,, TomTomTomTom BurdickBurdickBurdickBurdick1111,,,, LukeLukeLukeLuke ProProProPro1111,,,, WarrenWarrenWarrenWarren FriedlFriedlFriedlFriedl1111,,,, NickNickNickNick NovakNovakNovakNovak1111,,,, FengFengFengFeng QiuQiuQiuQiu2222,,,,JimJimJimJim B.B.B.B. McAlpineMcAlpineMcAlpineMcAlpine2222,,,, J.J.J.J. BrentBrentBrentBrent FriesenFriesenFriesenFriesen2,32,32,32,3,,,, andandandand GuidoGuidoGuidoGuido F.F.F.F. PauliPauliPauliPauli2222,,,,****

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

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

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

Enabling technology is the newly developed RT phase monitor, which utilizes a high-pressure, non-intrusivepermittivity 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 providesinput for volumetric and flow control regimes.(2) RT measurement of the partition-coefficient (K). This permits full normalization of the chromatographicx-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 thecomplete range of analytes.(3) RT mixing of biphasic solvent systems with a five-headed delivery system makes possible the directformulation 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 remoteinstrument operation, and is compatible with all major CS centrifugal column designs. Applications developed forthe targeted purification of botanical reference materials will be presented and demonstrate the power of RT controlin CS for natural products and pharmaceutical analysis.

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

targeted K-based analysis.


OOOO-2-2-2-2AAAA smallsmallsmallsmall volumevolumevolumevolume hydrostatichydrostatichydrostatichydrostatic CCCCCCCCCCCC columncolumncolumncolumn forforforfor fullfullfullfull andandandand quickquickquickquick solventsolventsolventsolvent selectionselectionselectionselection

AlainAlainAlainAlain Berthod,Berthod,Berthod,Berthod, KarineKarineKarineKarine Faure,Faure,Faure,Faure, JeremyJeremyJeremyJeremy Meucci,Meucci,Meucci,Meucci, NazimNazimNazimNazim MekaouiMekaouiMekaouiMekaouiInstitut des Sciences Analytiques, University of Lyon, Villeurbanne, France

*fax +33 472 448 319, e-mail: [email protected]

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

The two major problems in countercurrent chromatography (CCC) are first to find the appropriate biphasic liquidsystem for the desired purification and second to retain enough liquid stationary phase inside the CCC column so thatthe separation is doable. A new 38 mL volume hydrostatic CCC column is presented showing excellent liquidstationary phase retention even for the aqueous two-phase systems most difficult to work with. The small columnvolume 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 gainin experimental duration is obvious 12 min compared to 200 min (or 3 hours and 20 min). It is due first to the smallcolumn volume and second to the higher possible flow rate due to a better hold of the liquid stationary phase. Itsusefulness was demonstrated in the direct measurement of octanol/water partition coefficients.

TableTableTableTable 1.1.1.1. ComparingComparingComparingComparing characteristicscharacteristicscharacteristicscharacteristics ofofofof CCCCCCCCCCCC columnscolumnscolumnscolumns

SmallSmallSmallSmall CCCCCCCCCCCC RegularRegularRegularRegular CCCCCCCCCCCC

Column Vol. 38 mL 250 mL

Vs Sf=60% 23 mL 150 mL

Vr of K = 2 61 mL 400 mL

tr of K = 212 min @

5 mL/min

200 min @

2mL/min

Figure 1. Two hydrostatic CCC columns: left, the Kromaton FCPC250 that can hold different rotors including the 36 mL rotor; right,the Rousselet-Robatel-Decisive-Design prototype (R2D2), a 38 mLhydrostatic column. The small inset picture gives the prototypescale.

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 rotorminimizing size; the Rousselet-Robatel-Definitive-Design (R2D2) instrument contained the small hydrostatic rotorand 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 resultscan be easily transposed to bigger CCC columns of the hydrostatic as well as hydrodynamic type for larger loadsusing the same biphasic liquid system.


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



OOOO-3-3-3-3ConstructionConstructionConstructionConstruction ofofofof aaaa HSCCCHSCCCHSCCCHSCCC apparatusapparatusapparatusapparatus withwithwithwith columncolumncolumncolumn capacitycapacitycapacitycapacity ofofofof 12121212 orororor 15151515 litersliterslitersliters andandandanditsitsitsits applicationapplicationapplicationapplication asasasas flashflashflashflash countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography inininin quickquickquickquick preparationpreparationpreparationpreparation ofofofof

(-)-epicatechin(-)-epicatechin(-)-epicatechin(-)-epicatechin1111QingbaoQingbaoQingbaoQingbao Du,Du,Du,Du, 2222HeyuanHeyuanHeyuanHeyuan Jiang,Jiang,Jiang,Jiang, 1111，2222JunfengJunfengJunfengJunfeng Yin,Yin,Yin,Yin, 1111，2222YongquanYongquanYongquanYongquan Xu,Xu,Xu,Xu, 1111WenkaiWenkaiWenkaiWenkai Du,Du,Du,Du, 1111BoBoBoBo Li,Li,Li,Li, 1111QizhenQizhenQizhenQizhen Du*Du*Du*Du*

1Institute of Food Chemistry, Zhejiang Gongshang University, Hangzhou 3100125, China2 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:Keywords:Keywords: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, DuRecently suggested a term ‘‘Flash counter-current chromatography’’ (FCCC) which is defined as a real separationpossessing a characteristic that the flow rate of mobile phase (ml) is equal or greater than the square of the diameterof the column tubing (mm), i.e. Fc/d2≥1, here d and Fc are the diameter of the column tubing and the applied flow rateof mobile phase, respectively. FCCC separation finishes less than 5 h as the tubing length of column is less than 150m 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, symmetricallymounted, of 9-l capacity each which can be operated in parallel or series or as individual bobbins. This apparatus canbeen used for FCCC separation. However, in laboratory studies, HSCCC instruments with three columns in seriesoccupy more than 70% since successful commerce of Pharma Tech (USA) and Taotou (China). HSCCC instrumentswith 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 designstwo sets of columns with tubing bore of 10 mm and 12 mm yeilding 12 l and 15 l column capacity for a comparativeinvestigation of isolating (-)-epicatechin gallate (ECG) from tea extract by FCCC. Then, hydrolyzing the ECGfraction from FCCC for the separation of (-)-epicatechin (EC) by running flash column chromatography onmacroporous 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 countercurrentchromatography using a two-phase solvent system composed of hexane-ethyl acetate-water (1:2.5:4). In eachseparation, a sample amount of 200 g of the tea extract could be loaded to give 7.5 l of water solution containing 39 gof 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 ranges0.50-0.75. Under the same condition, the apparatus could load a sample amount of 200 g of the tea extract to yield9.0 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 flashcolumn 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.


OOOO-4-4-4-4SSSSeady-stateeady-stateeady-stateeady-state andandandand NNNNon-steadyon-steadyon-steadyon-steady statestatestatestate OOOOperationperationperationperation ofofofof CCCCounter-currentounter-currentounter-currentounter-current CCCChromatographyhromatographyhromatographyhromatography

devicesdevicesdevicesdevicesArtakArtakArtakArtak E.E.E.E. KostanyanKostanyanKostanyanKostanyan

Kurnakov Institute of General & Inorganic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 31, Moscow119991, Russia fax +495 9554834, e-mail: [email protected]

Keywords:Keywords:Keywords:Keywords: Counter-current chromatography; liquid–liquid extraction

Counter-current chromatography (CCC) is a non-steady state liquid–liquid extraction process. CCC apparatuses canbe operated under different conditions: in steady-state (as ultrahigh-efficient extraction columns for counter-currentseparation 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 purificationprocesses by using steady-state and non-steady state operating modes of CCC columns. Three approaches – the platetheory, the cell model and the Craig model, commonly used for mathematical description of separation processes inCCC and extraction columns, are discussed. The separation of the dual counter-current process conducted insteady-state (fractional extraction) and non-steady state (with impulse and non-impulse sample loading) operatingmodes are compared with the conventional CCC separation. In the dual counter-current separation, both phases aremobile and flowing in opposite directions in the CCC column; the sample is introduced at the middle of the columnand the components to be separated are eluted from opposite ends of the column. In steady-state (counter-currentfractional extraction) operating mode, where the sample is fed continuously at a constant rate, the concentration ofsolutes 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 ofsolutes eluted with phases as in case of conventional CCC separation are not constant. Basing on the cell modeltheory equations are presented allowing the simulation of the separation processes for both operating modes. Tocompare the steady-state extraction and chromatographic separation with impulse sample injection, a special case ofextraction, 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 xxxx and yyyy phases are determined by the following formulas:

1210 /)1(11

ϕϕϕ−+==

xxX

, ])1/[(1/

211

21

0 ϕϕϕ −+==

FFxyY

11

111

1 1 +

+

−−

= n

nn

AAA

ϕ , 12

122

2 1 +

+

−−

= n

nn

BBB

ϕ ,

DKFFA2

1= ,1

2

FKFB D= ,

10 FQx = ,

where F1 and F2 are the volumetric flow rates of xxxx and yyyy phases, respectively; KD = y/x is the partitioncoefficient; n1 and n2 are the numbers of equilibrium cells in the right and left of the column middle; Q is therate 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 conventionalCCC separation. The results obtained demonstrated the potential of the steady-state dual counter-currentprocesses for preparative and industrial-scale separations. Using CCC columns to conduct counter-currentextraction processes can allow increasing the productivity by an order of magnitude ensuring, a desirableseparation. In interaction with the suppliers, these devices are to be improved to provide a sound, stableoperation in the continuous steady-state conditions.



OOOO-5-5-5-5TTTTruerueruerue movingmovingmovingmoving bedbedbedbed CPCCPCCPCCPC forforforfor continuouscontinuouscontinuouscontinuous purificationpurificationpurificationpurification ofofofof naturalnaturalnaturalnatural substancessubstancessubstancessubstances

G.Audo*,G.Audo*,G.Audo*,G.Audo*, C.LeC.LeC.LeC.Le QuQuQuQuéééémeneurmeneurmeneurmeneur

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:Keywords:Keywords:Keywords: TMB, continuous,

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

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

Figure 1. True Moving Bed CPC general flowchart


OOOO-6-6-6-6DDDDesignesignesignesign ofofofof aaaa CCCContinuousontinuousontinuousontinuous CCCCentrifugalentrifugalentrifugalentrifugal PPPPartitionartitionartitionartition CCCChromatographichromatographichromatographichromatographic PPPProcessrocessrocessrocess

JohannesJohannesJohannesJohannes Goll,Goll,Goll,Goll, AndreasAndreasAndreasAndreas Frey,Frey,Frey,Frey, MirjanaMirjanaMirjanaMirjana Minceva*Minceva*Minceva*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:Keywords:Keywords:Keywords: sequential centrifugal partition chromatography, mathematical modeling, process design

Sequential centrifugal partition chromatography (sCPC) is a novel continuous cyclic separation process (1). UsingsCPC a mixture of components which distribute differently between the two phases of a biphasic liquid system canbe separated into two products. One of the specifics of the process is that the feed stream is introduced continuouslyin the middle of the sCPC unit whereas the two products are collected sequentially in each step of the cycle at theopposite ends of the unit. The two steps of one sCPC cycle differ by the liquid phase used as mobile phase (upper orlower phase) and its flow direction. The process includes several interconnected operating parameters (feedconcentration, feed and mobile phase flow rate in each step and the duration of each step of the cycle). Hence, theselection 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 todefine sCPC unit operating parameters which lead to complete feed separation, maximum productivity and minimumsolvent (mobile phase) consumption. The method includes several steps which are strongly supported bymathematical modeling and simulation.

At first, a biphasic solvent system from the ARIZONA solvent system family is selected by screening of the partitioncoefficients of the feed components using the a priori predictive thermodynamic method “Conductor-like ScreeningModel for Real Solvents” (COSMO-RS) (2). In the next step, the distribution equilibrium of the feed components inthe selected biphasic solvent system is determined by shake flask experiments. The maximum mobile phase flow ratein each sCPC step is derived from a study of the stationary phase retention as a function of the mobile phase flowrate. In the following, the column efficiency is determined by pulse injection experiments. The experimentallydetermined thermodynamic equilibrium, hydrodynamics and mass transfer parameters are incorporated in amathematical 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 ourprevious 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. Theproposed method is validated by comparison of the experimental and calculated sCPC separation performances. Incomparison with the conventional batch operation, the sCPC offers better

overall separation performances.

References1. 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.



OOOO-7-7-7-7IntermittentIntermittentIntermittentIntermittent Counter-currentCounter-currentCounter-currentCounter-current Extraction:Extraction:Extraction:Extraction: ScaleScaleScaleScale upupupup andandandand anananan ImprovedImprovedImprovedImproved BobbinBobbinBobbinBobbin

DesignDesignDesignDesignPeterPeterPeterPeter Hewitson*1,Hewitson*1,Hewitson*1,Hewitson*1, IanIanIanIan Sutherland1,Sutherland1,Sutherland1,Sutherland1, PhilipPhilipPhilipPhilip Wood2,Wood2,Wood2,Wood2, SvetlanaSvetlanaSvetlanaSvetlana Ignatova1Ignatova1Ignatova1Ignatova1

*1 Brunel Institute for Bioengineering, Brunel University, Uxbridge, UB8 3PH, UKTel: +44(0)1895 266917, Fax: +44(0)1895 274608, [email protected]

2 Dynamic Extractions, Ltd, Slough, UK

Keywords:Keywords:Keywords:Keywords: Intermittent counter-current extraction, liquid-liquid extraction

Intermittent counter-current extraction (ICcE) has been demonstrated as a potential liquid-liquid extraction methodfor continuous separation of compounds into two product streams and the isolation, concentration and enrichment ofa low abundance target compound, while washing away all other compounds (1). Further the scale-up of thetechnology using commercial twin-column CCC instruments has been successfully demonstrated (2) and the effect ofkey 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) topreparative DE- Midi instrument (912 mL) and on to pilot scale Maxi instrument (4.6 L) using a model mixture ofcompounds from the GUESSmix across a range of polarity hexane/ethyl acetate/methanol/water (HEMWat) phasesystems from a polar system (1:4:1:4) to a non polar system (4:1:4:1). These results are compared to a model ICcEto confirm the prediction of compound elution order.

With ICcE, the sample is continuously injected between the two columns while the mobile phase flow isintermittently switched between normal and reversed phase modes. When using conventional bobbins, with eachswitch an inherent delay occurs as a volume of the previous mobile phase is displaced from the flying leads into thefractions.

Therefore, a new bobbin design with modified end fittings which allow direct switching of mobile phase flow at thecolumn inlet is described (Figure 1). This design eliminates both the delay and the displaced dead volume whichusually occurs with each switch of mobile phase flow and therefore reduces any disruption to the column equilibriumas the previous mobile phase is pumped from the flying leads. These bobbins have been designed so they canoperate as standard isocratic columns, if required. Using the same GUESSmix and phase systems results shown a6% improvement in the retention times, equivalent to the eliminated delay. Further research is ongoing to assess theimprovements to the separation efficiency of these columns.

ReferencesReferencesReferencesReferences(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 2009200920092009, 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 2009200920092009, 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 2011201120112011, 1218, 6072-6078.

UPUPUPUPPumpPumpPumpPump

LPLPLPLPPumpPumpPumpPump

SamplePump

Column 1 Column 2

LP Detector

UP Detector

NORMAL PHASE

SamplePump

LP SampleLP SampleLP SampleLP Sample

UP SampleUP SampleUP SampleUP Sample

REVERSED PHASE

Figure 1: ICcE schematicshowing twin columns with separate in let and ou tlet leads for flow of upper and lower phases


OOOO-8-8-8-8SSSSelectionelectionelectionelection ofofofof thethethethe MMMMobileobileobileobile andandandand SSSStationarytationarytationarytationary PPPPhasehasehasehase inininin SSSSupportupportupportupport FFFFreereereeree liquid-liquidliquid-liquidliquid-liquidliquid-liquid

CCCChromatographyhromatographyhromatographyhromatography applyingapplyingapplyingapplying aaaa PPPPredicitveredicitveredicitveredicitve TTTThermodynamichermodynamichermodynamichermodynamic MMMModelodelodelodelAndreasAndreasAndreasAndreas Frey,Frey,Frey,Frey, MirjanaMirjanaMirjanaMirjana MincevaMincevaMincevaMinceva

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

Egerlandstraße 3, 91058 ErlangenFax: 09131-85-27441

E-Mail: [email protected]*[email protected]

Keywords:Keywords:Keywords: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 toa selection of a biphasic solvent system. The limitless choice of solvents requires a systematic method for theselection of the appropriate solvent combination for a given separation task. At the moment, this selection isperformed by “trial and error” experimental screening procedures, using only a restricted number of solvents andsolvent combinations.In this work a general approach for the selection of the mobile and the stationary phase for a specific LLC separationtask 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 targetsolutes 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 phasecompositions and diagrams. In a next step, a screening of the pre-selected biphasic liquid systems is performed usingthe 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 furtherevaluated 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 solventconsumption is selected for the given separation task.It is demonstrated that with the proposed approach the extensive experimental effort needed for the selection of thebiphasic liquid system can be significantly reduced.


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

mailto:[email protected]*



OOOO-9-9-9-9SelectionSelectionSelectionSelection andandandand useuseuseuse ofofofof aqueousaqueousaqueousaqueous twotwotwotwo phasephasephasephase systemssystemssystemssystems inininin centrifugalcentrifugalcentrifugalcentrifugal partitionpartitionpartitionpartition

chromatographychromatographychromatographychromatographyChristophChristophChristophChristoph Schwienheer*,Schwienheer*,Schwienheer*,Schwienheer*, StephanStephanStephanStephan Adelmann,Adelmann,Adelmann,Adelmann, JulianeJulianeJulianeJuliane Merz,Merz,Merz,Merz, GerhardGerhardGerhardGerhard

SchembeckerSchembeckerSchembeckerSchembecker

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 thehydrodynamics in the chambers. Especially flow pattern and the resulting stationary phase hold-up andinterfacial area for mass transfer are important parameters. While there are already some experimental resultsfor aqueous organic systems and some hints on appropriate selection of phase system conditions and operationparameters [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 inadequate concentrations. ATPS are gentle for the separation of biotechnological products like proteins orantibodies. Resulting on their physical properties (low interfacial tension, low density difference and highviscosities compared to usual aqueous organic solvent systems) the phase separation can only be achievedpoorly. 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 andinterfacial tension) were measured by varying the molecular weight of polyethylenglycol (PEG) and theconcentrations of PEG and phosphate salt. The viscosity increases more with increasing molecular weight thanwith increasing the concentration of PEG. The density difference and interfacial tension, however, are mainlyincreased by increasing concentrations of the phase forming components.Based on these results several ATPS were selected for visualization of their hydrodynamical behavior in CPCchambers. Using an optical experimental setup [2] and a phase selective dye for coloring the mobile phase wecould observe the flow of the mobile phase and measure the hold up of stationary phase for different operatingparameter. As a result of these investigations, the hydrodynamical ability of different ATPS could bedetermined and appropriate operation parameter could be found. In general, the lower viscous phase of thesystem should be the mobile phase and the rotational speed of the CPC should be as high as possible. Asexemplarily shown in figure 1 it is useful to increase the concentrations of the phase forming components todecrease the bleeding of stationary phase and achieve a higher hold up. Finally, separation experiments wereperformed for a mixture of exemplary proteins to evaluate the influence of mentioned parameters on theseparation efficiency.


1. L. Marchal et.al; AIChE Journal; vol. 48, no. 8; 2002; 1692-17042. S. Adelmann et.al; Journal of Chromatography A; vol. 1218, no. 32; 2011; 5401-54133. I. Sutherland et.al; Journal of Chromatography A; vol. 1218, no. 32; 2011; 5527-5530

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.


OOOO-10-10-10-10MMMModellingodellingodellingodelling CCCCounter-currentounter-currentounter-currentounter-current CCCChromatographyhromatographyhromatographyhromatography usingusingusingusing aaaa TTTTemperatureemperatureemperatureemperature DDDDependenceependenceependenceependence

PPPPlatelatelatelate MMMModelodelodelodelYunYunYunYun WeWeWeWeiiii *,*,*,*, FengkangFengkangFengkangFengkang Wang,Wang,Wang,Wang, ShuiShuiShuiShui WangWangWangWang

*State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 15Beisanhuan East Road, Chaoyang District, Beijing 100029, China.Tel & Fax: 0086 10 64442928. E-mail: [email protected]

Keywords:Keywords:Keywords:Keywords: Counter-current Chromatography, partition coefficient K values, temperature, modelling

ABSTRACTABSTRACTABSTRACTABSTRACTRecently research workers have built several math models of counter-current chromatography (CCC), such as cellmodel1, CCC distribution model2, Continuous-Stirred Tank Reactors model3, probability model4. A new math modelof 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 mathmodelling of the partition action of CCC can be achieved by using Matlab to do computer programming. Therelationship 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. Rvalues are related to K values, as a result, temperature change finally leads to resolutions between differentcompounds change. K values at different temperatures of a certain compound in a given system were calculatedaccording to those equations above. New program with temperature parameter was applied in prediction ofexperimental result, which decreased the error in modelling prediction. The retention time error is 9.22% for peak 2and 5.46% for peak 4 (fig.1), respectively, without considering temperature effect. When temperature was taken intoconsideration, the error was significantly reduced (fig.2).

This temperature dependence plate model can well explain why CCC partition efficiency is not the same in differentseasons without temperature controller, and it can be more accurate in CCC separation prediction than previousmodels. As we all know, two compounds can’t be separated when the separation factor is 1. However, if theirlogK-1/T equations are different, separation can be achieved by changing temperature and the optimal temperaturecan be obtained from this new model. Those logK-1/T equations may be applied to other models in the future.

ReferencesReferencesReferencesReferences1. Kostanian, A. E. J. Chromatogr. A 2002200220022002, 973, 39-46.2. Sutherland, I. A.; Folter, J. de. ; Wood, P. J. Liq. Chromatogr. Related Technol. 2003200320032003, 26, 1449-1474.3. Guzlek, H.; Baptista, I.I.R.; Wood P. L.; Livingston, A. J. Chromatogr. A, 2010201020102010, 1217, 6230–6240.4. Folter, J. d.; Sutherland I. A. J. Chromatogr. A, 2011201120112011, 1218, 6009-6014.

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 7.00.000

0.005

0.010

0.015

0.020

0.025

Co

nc(

mg

/mL

)/N

orm

alis

ed

Ab

sorb

an

ce

t /h

experim en ta l resu lt s im u la ted 2 s im u la ted 4

24

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 .00 .0 0 0

0 .0 0 5

0 .0 1 0

0 .0 1 5

0 .0 2 0

0 .0 2 5

Co

nc(

mg

/mL

)/N

orm

alis

ed

Ab

sorb

an

ce

t/h

e xp e rim e n ta l re su lt s im u la te d 2 s im u la te d 4

24

Fig.1. Black line: chromatogram of the crude extract fromFlaveria bidentis (L.) Kuntze by HSCCC. Solvent system: ethylacetate–methanol–water 50:1:50 (v/v); stationary phase: upperorganic phase, mobile phase: lower aqueous phase, flow rate:2.0 ml/min, revolution speed: 800 rpm, sample: 400mgdissolved in 10 ml lower phase. Peaks: 2 waspatuletin-3-O-glucoside, 4 was astragalin. Red line:simulated peak 2 not considering temperature effectBlue line: simulated peak 4 not considering temperature effec

Fig.2. Black line: experimental result

Red line: simulated peak 2 at 291.15K \

Blue line: simulated peak 4 at 296.35K


app:ds:introduce

app:ds:previous

app:ds:optimal


OOOO----11111111MMMModelingodelingodelingodeling GGGGradientradientradientradient EEEElutionlutionlutionlution inininin CCC:CCC:CCC:CCC: EEEEfficientfficientfficientfficient SSSSeparationeparationeparationeparation ofofofof TTTTanshinonesanshinonesanshinonesanshinones

S.S.S.S. Wu1Wu1Wu1Wu1 D.D.D.D. Wu1Wu1Wu1Wu1 J.Liang1J.Liang1J.Liang1J.Liang1 A.A.A.A. Berthod2Berthod2Berthod2Berthod21-Siyuan research center in Pharmacy and Biotoxicology, College of Life Science, Zhejiang University,

Hangzhou 310058, China2-Institut of Analytical Sciences, CNRS, University of Lyon, 69622 Villeurbanne, France

*fax +33 472 448 319; e-mail: [email protected]

Keywords:Keywords:Keywords:Keywords: Liquid systems, gradient elution, theory, tanshinones

Counter-current chromatography (CCC) is a support-free liquid-liquid chromatography using centrifugal fieldsto hold the liquid stationary phase. CCC has been widely applied in the separation of various natural and syntheticcomponents using a variety of biphasic liquid systems. The related hexane or heptane/ethyl acetate/methanol orethanol/water biphasic liquid systems (HEMWat or HEEWat systems, respectively) demonstrated their significancein CCC.

Gradient is difficult in CCC since any composition change in one phase induces a composition change of theother phase to maintain phase equilibrium. This work provides a new insight into linear gradient elution in CCCthat is feasible with some biphasic liquid systems such as selected compositions of the HEMWat or HEEWatsystems.

Two approaches are presented and compared: the first approach proposes the equations modeling solutemotion inside the CCC column; the second approaches consider the gradient mobile phase composition change usinga 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:Watcompositions with E+Wat = 10, were experimentally studied from Wat = 1 to Wat = 9. They showed moderatechanges in the upper organic phase compositions. The two model are tested with the separation of tanshinones fromthe rhizome of Salvia miltiorrhiza Bunge.

Different linear solvent gradient profiles were experimentally performed between 8:2:5:5 and 8:2:3:7 HEEWatcompositions and the results were evaluated using the two proposed models. Five tanshinones includingdihydrotanshinone I, cryptotanshinone, tanshinone I, 1,2-dihydro tanshinquinone and tanshinone IIA have beensuccessfully separated (>95% purities). The gradient models can be used only with biphasic liquid systems inwhich one phase shows minimum composition changes when the other phase composition changes notably. Thiscase is not the general case for biphasic liquid systems but can be applied with specific compositions of thequaternary HEMWat or HEEWat most useful CCC liquid systems.

0 70 140 210 280 350 420 480

Time (min) 0 70 140 210 280 350 420

1111

1111

2222

2222

3333

3333

4444

4444

5555

5555

AAAA

BBBB

UV absorbance 220 nm (a.u.)

280 nm

FigureFigureFigureFigure 1111: Gradient CCC separations a crude

extract of S. miltiorrhiza Bunge. The separation

is done with the HEEWat solvent system 8:2:5:5

for AAAA: 250 min and BBBB: 200 min and next, the

mobile phase is linearly changed to the lower

aqueous phase of the 8:2:7:3 HEEWat system in

AAAA: 150 min and BBBB: 100 min. Compounds 1111,

dihydrotanshinone I; 2222, cryptotanshinone; 3333,

tanshinone I; 4444, 1,2-dihydrotanshinquinone; 5555,

tanshinone IIA. CCC conditions: flow rate: 2

mL/min; rotation speed: 850 rpm; temperature:

30oC; sample loading: 200 mg injected in 10 mL;

elution mode: head-to-tail elution direction. Initial



OOOO----11112222InvestigationInvestigationInvestigationInvestigation ofofofof hydrodynamicshydrodynamicshydrodynamicshydrodynamics inininin CPCCPCCPCCPC chamberschamberschamberschambers usingusingusingusing imageimageimageimage processingprocessingprocessingprocessing andandandand

computationalcomputationalcomputationalcomputational fluidfluidfluidfluid dynamicsdynamicsdynamicsdynamicsJulianeJulianeJulianeJuliane Merz*,Merz*,Merz*,Merz*, ChristophChristophChristophChristoph Schwienheer,Schwienheer,Schwienheer,Schwienheer, StephanStephanStephanStephan Adelmann,Adelmann,Adelmann,Adelmann, GerhardGerhardGerhardGerhard

SchembeckerSchembeckerSchembeckerSchembecker

TU Dortmund, Laboratory of Plant and Process Design

* Emil-Figge-Straße 70, 44227 Dortmund, Germany; fax +492317552341; [email protected]

Keywords:Keywords:Keywords:Keywords: CPC, flow pattern, operating parameter

Centrifugal partition chromatography (CPC) is a kind of liquid-liquid partition chromatography in which theseparation mechanism is based on the principle of different distribution of components between two immiscibleliquids. 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 stronglyinfluenced by the hydrodynamics in the chambers. Especially flow pattern and the resulting stationary phase hold-upand interfacial area for mass transfer are important parameters. Their relation on physical properties of solventsystems, operating parameters and chamber geometry are not completely understood yet. Only a few research groupshave developed a possibility to observe the flow of mobile phase through the stationary phase in the rotatingchambers [1].

In order to receive a more detailed impression of flow pattern and derived parameters like stationary phase hold-upand interfacial surface area, we have developed a new method for flow visualization. The developed opticalmeasurement device is attached to a conventional FCPC® unit by Kromaton. We use a single disc covered by acrylicglass, in order to look inside the chambers. To make the flow of mobile phase visible, it is colored with a phaseselective dye. The dye absorbs the light of a LED installed at the bottom of the apparatus and pictures were taken bya monochromatic CCD camera installed at the top (see figure 1). Both, LED and camera are triggered by a fork lightbarrier, to achieve one picture per revolution. After some image processing using customized programs, high contrastimages 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 phasesystems 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 mobilephase and the resulting hold up of stationary phase have to be optimized. Additionally, new CPC chambers for moreefficient separations are investigated by using the optical measurement device and additionally computational fluiddynamics as presented by Adelmann et.al. [3].

Figure 1. Sketch of the optical measurement device setup.


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


OOOO----11113333RRRResearchesearchesearchesearch ofofofof IIIInteractionnteractionnteractionnteraction betweenbetweenbetweenbetween IIIIoniconiconiconic LLLLiquidiquidiquidiquid andandandand TTTTeaeaeaea PPPPolyphinolsolyphinolsolyphinolsolyphinols bybybyby computercomputercomputercomputer

simulationsimulationsimulationsimulation assistedassistedassistedassisted HHHHigh-performanceigh-performanceigh-performanceigh-performance CCCCounter-currentounter-currentounter-currentounter-current CCCChromatographyhromatographyhromatographyhromatographyChenChenChenChen Xiao-FenXiao-FenXiao-FenXiao-Fen1,21,21,21,2,,,, HuangHuangHuangHuang Xin-YiXin-YiXin-YiXin-Yi1111,,,, WangWangWangWang Gao-hongGao-hongGao-hongGao-hong1,21,21,21,2,,,, ZhangZhangZhangZhang JiaJiaJiaJia1111,,,, DiDiDiDi Duo-LongDuo-LongDuo-LongDuo-Long1111****

1 Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for Natural Medicine of GansuProvince, 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]

KeywordsKeywordsKeywordsKeywords: 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 environmentalbenefits, nonvolatility, noninflammability, high thermal stability and designability. In recent years, it seemed to ILs inhigh-speed counter-current chromatography (HSCCC) has attracted scientists’ attention. However, to date, there areno 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 similarstructure. In this study, they were chosen as model moleculars to study the effect of ionic liquid as solvent additivesin HPCCC separation.

Basic solvent system of ethyl acetate- water (5:5, v/v) was employed. It was found that three target compoundswere inclined to distribute in upper phase. As the increase of hydrophilic ionic liquids added in solvent system, theywere draw to lower phase due to the attraction of ionic liquids, which indicated by the decrease of K values (Figure2). It was planned to combine computer simulation and spectroscopy methods with HPCCC to illustrate theinteraction between ionic liquids and target compounds.

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

Figure 1. Structures of

polyphenols, A: EGCG, B:

ECG, C: GCG.

Figure 2. The effect of the concentration

of 1-methyl-3-methylimidazolium

tetrafluorobarate on K-values in ethylacetate–water (5:5, v/v).


app:lj:%E5%8F%AF%E8%AE%BE%E8%AE%A1%E6%80%A7?ljtype=blng&ljblngcont=0&ljtran=designability


OOOO----11114444ModelingModelingModelingModeling ofofofof stepstepstepstep wisewisewisewise gradientsgradientsgradientsgradients inininin HSCCC;HSCCC;HSCCC;HSCCC; AAAA rapidrapidrapidrapid andandandand economicaleconomicaleconomicaleconomical strategystrategystrategystrategy forforforfor

oneoneoneone stepstepstepstep separationseparationseparationseparation ofofofof eighteighteighteight dihydropyranocoumarinsdihydropyranocoumarinsdihydropyranocoumarinsdihydropyranocoumarins fromfromfromfrom SeseliSeseliSeseliSeseli resinousmresinousmresinousmresinousm....

OmerOmerOmerOmer ShehzadShehzadShehzadShehzad1111,,,, SalmanSalmanSalmanSalman KhanKhanKhanKhan1111,,,, InInInIn JinJinJinJin HaHaHaHa1111,,,, KyoungKyoungKyoungKyoung JinJinJinJin LeeLeeLeeLee1111,,,, AlevAlevAlevAlev TosunTosunTosunTosun2222,,,, YeongYeongYeongYeong ShikShikShikShik KimKimKimKim1111****

1Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul151-742, 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:Keywords:Keywords: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 bigchallenge for counter-current chromatography (CCC). Once a suitable solvent system is selected, the separationprocess 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 gradientsfor quick isolation of eight dihydropyranocoumarins from the hexane fraction of Seseli resinosum in a singlerun. For ensuring the high purity of each compound only the main peaks were collected while all the shoulderpeaks 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 preparedusing 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, thatlead to decrease in operation time and increase in stationary phase retention. This separation process producedeight 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 allowus to generate a strategy for target separation of compounds with a broad range polarity from any naturalproduct.


OOOO-15-15-15-15IsolationIsolationIsolationIsolation ooooffff bioactivebioactivebioactivebioactive polyphenolspolyphenolspolyphenolspolyphenols ffffromromromrom bbbby-productsy-productsy-productsy-products ooooffff foodfoodfoodfood processingprocessingprocessingprocessing usingusingusingusing

countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography

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

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

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

*Correspondence address marked with asterisk

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

Strategies for the large-scale isolation and fractionation of polyphenolic extracts from waste material will bedemonstrated. Examples are the preparation of bioactive by-products from side streams of the wine and fruit juiceindustry (1,2). The combination of countercurrent chromato-graphy with membrane techniques allows the separationof 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 fractionfree of other copigments and polymeric phenols can be obtained (3). For further isolation of bioactives from thecopigment fraction, countercurrent chromatography is applied. Techniques that have been applied include inter alialow speed rotary countercurrent chromatography (LSRCCC) and spiral-coil low speed rotary countercurrentchromatography (Spiral-coil LSRCCC). With regards to the polymeric constituents, especially the procyanidines, adepolymerization process will be presented converting the high molecular compounds in bioavailable ones (dimersand trimers) (4-6). Finally initial results concerning the use of grape vine shoots for the preparation of highlybioactive oligomeric stilbenes and their subsequent separation using countercurrent chromatography will bepresented (7). These examples demonstrate the need for preparative CCC-systems in order to supply the bioactives insufficient amounts for the food and pharmaceutical industry.


1. M. Schwarz, S. Hillebrand, S. Habben, A. Degenhardt, P. Winterhalter, Application of high-speed countercurrentchromatography to the large-scale isolation of anthocyanins. Biochem. Engineering J. 14141414, 179-189 (2003).

2. D. Cooke , M. Schwarz, D. Boocock, P. Winterhalter, W.P. Steward, A.J. Gescher, T.H. Marczylo, Effect ofcyanidin-3-glucoside and an anthocyanin mixture from bilberry on adenoma development in the ApcMin mouse model ofintestinal carcinogenesis - Relationship with tissue anthocyanin levels. Int. J. Cancer 119119119119, 2213-2220 (2006).

3. A. Juadjur, P. Winterhalter, Development of a novel adsorptive membrane chromatographic method for the fractionation ofpolyphenols from bilberry. J. Agric. Food Chem. 60606060, 2427-2433 (2012).

4. N. Köhler, V. Wray, P. Winterhalter, Preparative isolation of procyanidins from grape seed extracts by high-speedcounter-current chromatography. J. Chromatogr. A 1177117711771177, 114-125 (2008).

5. T. Esatbeyoglu, P. Winterhalter, Preparation of dimeric procyanidins B1, B2, B5, and B7 from a polymeric procyanidinfraction of black chokeberry (Aronia melanocarpa). J. Agric. Food Chem. 58585858, 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. 58585858,7820-7830 (2010).

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

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


OOOO-16-16-16-16StudyStudyStudyStudy onononon preparativepreparativepreparativepreparative separationseparationseparationseparation andandandand purificationpurificationpurificationpurification ofofofof chemicalchemicalchemicalchemical compoundscompoundscompoundscompounds fromfromfromfrom

plantsplantsplantsplants bybybyby hsccchsccchsccchsccc

ChenChenChenChen Xiao-Fen1,2,Xiao-Fen1,2,Xiao-Fen1,2,Xiao-Fen1,2, HuangHuangHuangHuang Xin-Yi1,Xin-Yi1,Xin-Yi1,Xin-Yi1, WangWangWangWang Xiao-fei1,Xiao-fei1,Xiao-fei1,Xiao-fei1, ShiShiShiShi Ming-rui1,Ming-rui1,Ming-rui1,Ming-rui1, WangWangWangWang Gao-hong1,2,Gao-hong1,2,Gao-hong1,2,Gao-hong1,2,ZhangZhangZhangZhang Jia1,3,Jia1,3,Jia1,3,Jia1,3, DiDiDiDi Duo-Long1,3*Duo-Long1,3*Duo-Long1,3*Duo-Long1,3*

1Key laboratory of Chemistry of Northwestern Plant Resources, Chinese Academy of Sciences & Key Laboratory forNatural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy ofSciences, Lanzhou

7300002Graduate University of Chinese Academy of Sciences, Beijing 100049

3Department of Pharmacy，Gansu College of Traditional Chinese Medicine，Lanzhou 730000*Corresponding author fax: 0931-4968094; e-mail: [email protected]

Keywords:Keywords:Keywords: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 thepreparative separation and purification of chemical components from plants due to its unique advantages. In thispaper, advancements on application of HSCCC in separation and purification of active compounds from plants arepresent by our research group as follows:

1. Preparative separation and purification of highlypolarity and thermolabile components from the rhizome ofSphallerocarpus 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 ofRadix Astragali by HSCCC, respectively.

3. New Way development: Online extraction and .preparative separation of chemical constituents from Brassicanapus 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 ofchemical components from Brassica napus L. pollen and Tea.

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

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



OOOO-17-17-17-17EEEEnantioseparationnantioseparationnantioseparationnantioseparation ofofofof 2-phenylpropionic2-phenylpropionic2-phenylpropionic2-phenylpropionic acidacidacidacid bybybyby recyclingrecyclingrecyclingrecycling highhighhighhigh speedspeedspeedspeed counter-counter-counter-counter-

currentcurrentcurrentcurrent chromatographychromatographychromatographychromatography usingusingusingusing hydroxypropyl-hydroxypropyl-hydroxypropyl-hydroxypropyl-ββββ-cyclodextrin-cyclodextrin-cyclodextrin-cyclodextrin asasasas chiralchiralchiralchiral selectorselectorselectorselectorYeYeYeYe ZhengZhengZhengZheng，ShengqiangShengqiangShengqiangShengqiang Tong*Tong*Tong*Tong*，JizhongJizhongJizhongJizhong YanYanYanYan

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:Keywords: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 of2-phenylpropionic acid enantiomer using hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector. The two-phasesolvent 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 ofHP-β-CD, pH value of the aqueous phase, the separation temperature. Suitable elution mode was selected forHSCCC 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. Thepurities of both of the fractions including (+)-2-phenylpropionic acid and (-)-2-phenylpropionic acid from thepreparative 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: solventsystem: 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 theaqueous phase; stationary phase: upper organic phase; mobile phase: lower aqueous phase; sample solution: 50 mg of 2-phenylpropionic acidracemate 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 HSCCCfraction containing (+)-2- phenylpropionic acid; (c) preparative HSCCC fraction containing (-)-2- phenylpropionic acid.


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


OOOO-18-18-18-18ExcellentExcellentExcellentExcellent fractionfractionfractionfraction effecteffecteffecteffect ofofofof HSCCCHSCCCHSCCCHSCCC onononon thethethethe completedcompletedcompletedcompleted separationseparationseparationseparation ofofofof amideamideamideamide

compoundscompoundscompoundscompounds inininin blackblackblackblack pepperpepperpepperpepperYongyangYongyangYongyangYongyang Jin,Jin,Jin,Jin, DengyongDengyongDengyongDengyong Qian,Qian,Qian,Qian, QizhenQizhenQizhenQizhen Du*Du*Du*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:Keywords:Keywords: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 aspice and also as a folk medicine. Black pepper, the fruit of Piper nigrum L. is widely used as an anodyne and atreatment for stomach disease in China. A lot of works on Piper species showed that the major bioactive componentsare amides. The preparative separation and purification of amides from plant materials by conventional methods istedious and usually requires multiple chromatography steps, such as column chromatography (CC) and thin-layerchromatography (TLC). It is very difficult to obtain high pure amides using traditional chromatography since amidescan 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 noadsorptive sample loss and deactivation, tailing of solute peaks, contamination and etc. Thus, it is a best way tofractionate crude plant extract for separation and preparation of the whole components in the extract. In the presentstudy, we achieved an excellent fractionation of amides in the crude extract of the fruit of Piper nigrum L., andobtained 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 wascollected by a fraction collector.

The high-speed countercurrent chromatograph used in the present study was constructed at the Institute of Foodand Biological Engineering, Zhejiang Gongshang University, Hangzhou, China. The apparatus was equipped with a1200-ml column with 6-layer coils made of a 5.0 mm i.d. Teflon tubing. For the separation a K-1800 Wellchrompreparative HPLC pump (Knauer, Germany), a 100 ml sample loop made of 3 mm i.d. Teflon tubing, and a B-684collector (Büchi, Switzerland) with 15-ml tube racks was used. The separation procedure began from the filling thecolumn with the stationary phase. Then the apparatus was rotated at 1000 rpm and the sample solution (3.5 g crudeamide components in 100 ml mobile phase) was injected into the CCC system through the Teflon sample loop withthe 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 separationafford to 7 fractions: FrA to FrG. Concentration and dryness in vacuum of FrD, FrE and FrF resulted in compounds10101010 (135 mg), 11111111 (85 mg) and 12121212 (265 mg).

Preparative HPLC of FrA (100 mg) afforded compounds 1111 (46 mg) and 2222 (41 mg) while FrB (100 mg) gavecompounds 3333 (13 mg), 4444 (15 mg), 5555 (28 mg) and 6666 (31 mg); FrC (100 mg) afforded compounds 7777 (33 mg), 8888 (14 mg)and 9999 (17 mg); and FrG (from stationary phase) (100 mg) yielded compounds 13131313 (74 mg).

Fig. 1 TLC-chromatogram of 3.5 g of the crude amide compounds from black pepper. Solvent system: n-hexane-ethylacetate-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); colorizingreagent: 10% sulfuric acid-ethanol solution.

ESI-MS and NMR analysis indicated the 12 compounds were retrofractamide-A (1111),(2E,4E)-N-isobutyl-decadienamide (2222), retrofractamide-C (3333), piperoleine A (4444), dehydropiperoleine A (5555),retrofractamide-B (6666), piperoleine B (7777), pipernonaline (8888), pipercyclobutanamides A (9999), dehydropipernonaline (10101010),piperine (11111111) , (2E,4E,12Z)-N-isobutyl-octadecatrienamide (12121212), and (2E,4E,14Z)-N-isobutyleicosa-2,4,14-trienamide (13131313).

Result of the present study exhibits that HSCCC possesses excellent fraction effect for completed separation ofnatural 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.


OOOO-19-19-19-19MultifunctionalMultifunctionalMultifunctionalMultifunctional tetranortriterpenoidstetranortriterpenoidstetranortriterpenoidstetranortriterpenoids fromfromfromfrom AzadirachtaAzadirachtaAzadirachtaAzadirachta indica:indica:indica:indica: aaaa separationseparationseparationseparation

challengechallengechallengechallenge solvedsolvedsolvedsolved bybybyby MLCCCMLCCCMLCCCMLCCC atatatat bothbothbothboth thethethethe analyticalanalyticalanalyticalanalytical andandandand preparativepreparativepreparativepreparative scalescalescalescaleHansHansHansHans E.E.E.E. Hummel*1,Hummel*1,Hummel*1,Hummel*1, 2,2,2,2, D.D.D.D. F.F.F.F. Hein1,Hein1,Hein1,Hein1, Y.Y.Y.Y. Ma3,Ma3,Ma3,Ma3, Y.Y.Y.Y. Ito3Ito3Ito3Ito3

*1Chair of Organic Agriculture, Justus-Liebig-University Giessen, Germany. [email protected] Natural History Survey, Prairie Research Institute, University of Illinois, Champaign, USA

3Laboratory of Biophysical Chemistry, N.I.H., NHLB Institute, Bethesda, Md. 20892 –USAKeywords:Keywords:Keywords:Keywords: Azadirachta indica, A. excelsa, azadirachtin, biopesticides, organic agriculture

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

A series of solvent extraction steps of crushed neem and marrango seed kernels [1-3] yielded defatted "AZT" extractscontaining 6-12% of tetranortriterpenoids. This was the starting material for various purification attempts using thefollowing 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 MLCCCruns, typically with intended overloading, resulted in aza purities between 70 and 90 % and had to be repeated by asubsequent run on the triplet CCC-1000 coil with 180 ml volume. Typical solvent systems used consisted of biphasicmixtures of hexane: EtOAc:CH3OH:H2O = 4:5:4:5 and 3:5:3:5. Monitoring was accomplished by HPLC with onlineUV 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:sulfuricacid:anisaldehyde reagent 25:1:0.1 and heating for 1 to 2 minutes at 140°C. Purities of products after 2 to 3 runs wereabove 97% and suitable for NMR as well as biological testing with the Epilachna varivestis and Spodoptera littoralistest 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 thetwo substances. The first had a final HPLC purity of 99.6%. After several weeks in which the solvents could slowlyevaporate, spontaneous crystallization of aza occurred and produced tetragonal crystals. This observation providesadditional 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. NeemConference (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.


OOOO-20-20-20-20BioassayBioassayBioassayBioassay guidedguidedguidedguided separationseparationseparationseparation ofofofof bioactivebioactivebioactivebioactive componentscomponentscomponentscomponents inininin naturalnaturalnaturalnatural productsproductsproductsproducts viaviaviavia

counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography coupledcoupledcoupledcoupled withwithwithwith LC/MS/MSLC/MS/MSLC/MS/MSLC/MS/MSRuilinRuilinRuilinRuilin Hu,Hu,Hu,Hu, PengPengPengPeng Chen,Chen,Chen,Chen, YuanjiangYuanjiangYuanjiangYuanjiang Pan*Pan*Pan*Pan*

Department of Chemistry, Zhejiang University, Hangzhou 310027, China*Correspondence address: fax: +86 571 87951629. Email address: [email protected]

Keywords:Keywords:Keywords: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 pharmaceuticalindustries, despite the remarkable progress in synthetic organic chemistry of the twentieth century, over 25% ofprescribed medicines in industrialized countries derive directly or indirectly from plants. This percentage can reach50% when the over-the-counter (OTC) market is taken into consideration. It’s of great importance to separate thebioactive compounds in medicinal plants with high efficiency. However, the bioactive compounds may be lost duringthe multiple sample preparation processes when using solid-phase supporting materials. Therefore, it’s better to avoidirreversible adsorption in whole separation procedure.

Counter-current chromatography (CCC) uses two immiscible solvents without using solid matrix. The liquidstationary phase is retained by the centrifugal field. The mobile phase continuously passes through the stationaryphase to achieve efficient partitions. Since CCC first developed by Ito, it has been widely considered as an efficientseparation tool in natural products separation. The crude medicinal plant extract contains various compoundsdiffering in concentrations, polarities, molecular weights and bioactivities. Rapid identification and separation of thebioactive compounds from the crude natural plant extracts inspires great interests in recent years. The extrusion CCCmethod (EECCC&BECCC) has been proved as an efficient fractionation method which may replace traditionalfractionation procedure [1, 2]. The hydrophilic and hydrophobic components can be well separated by extrusionmethod. Moreover, due to the extrusion step, it saves a lot of time and solvents. On-line bioassay coupled with HPLChas 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 methodstogether and applied in natural drug discovery program. The strategy of separation of the bioactive compounds is asfollows: In the first step, the extrusion method was applied to rapid fractionating of the crude extract to obtain severalfractions. 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. Inthe third step, the target compounds were further isolated by CCC. The advantage of this strategy compared withtraditional method used in natural drug discovery program is obvious: The bioactive compounds can be isolated inshort analysis time and the minor components won’t be lost in the screen process. Two natural productsAmpelopsis heterophylla and Vitis wilsonae Veitch were evaluated by this strategy. The results indicated that thisstrategy had great potential to be applied in natural drug discovery program.

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


1. Y. Lu, A. Berthod, R. Hu, W. Ma, Y. Pan, Anal. Chem. 2009,,,, 81, 40482. 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.



OOOO----22221111SeparationSeparationSeparationSeparation andandandand purificationpurificationpurificationpurification ofofofof quanternaryquanternaryquanternaryquanternary protoberberineprotoberberineprotoberberineprotoberberine alkaloidsalkaloidsalkaloidsalkaloids fromfromfromfrom

CorydalisCorydalisCorydalisCorydalis saxicolasaxicolasaxicolasaxicola BuntingBuntingBuntingBunting bybybyby HSCCCHSCCCHSCCCHSCCCZhichaoZhichaoZhichaoZhichao He1He1He1He1,2,2,2,2,,,, QiongxianQiongxianQiongxianQiongxian Ye1,Ye1,Ye1,Ye1, JunyanJunyanJunyanJunyan Wu2,Wu2,Wu2,Wu2, DepoDepoDepoDepo YangYangYangYang1111,,,, LeslieLeslieLeslieLeslie BrownBrownBrownBrown3,3,3,3, LinLinLinLin

JiangJiangJiangJiang1111,,,, LongpingLongpingLongpingLongping Zhu1,Zhu1,Zhu1,Zhu1, DongmeiDongmeiDongmeiDongmei Wang1*Wang1*Wang1*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:Keywords:Keywords:Keywords: Corydalis saxicola, alkaloids, HSCCC

Corydalis saxicola Bunting, a well-known Chinese herbal medicine, possesses antibacterial, antiviral, andhepatoprotective activities, etc. The active constituents were reported to be a series of tertiary and quaternaryprotoberberine alkaloids [1,2]. In this study, a HSCCC method for rapid separation of three main active quaternaryprotoberberine alkaloids was established. After partitioned by different organic solvents, the n-butanol extract of theethanolic 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 waterlayer mainly contained dehydrocheilanthifo-line (1111), dehdroapocavidine (2222), dehydrocavidne (3333) and cavidine (4444)(Fig. 1B) was selected for separation on HSCCC. Screening the K values (data not shown) of the four compounds inten 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) showedthat the four target compounds might be separated on HSCCC. however, cavidine (4444) was co-eluted with the otherthree compounds unexpectively on real HSCCC separation. In order to solve this problem, buffer solution withdifferent pH values was used instead of ammonia in Solvent I, and cavidine could be removed when pH of buffersolution was 9.0 (Solvent II). The result in Fig.2A indicated that peak A1 contained componds 1-31-31-31-3, and cavidine wasobtained in peak A2. The peak A1 was further separated on HSCCC by Solvent I. As shown in Fig.2B, peak B1 (3333),peak B2 (2222) and peak B3 (1111) with purities over 95% were obtained, respectively.

Figure 1. Chromatograms of n-butanol extract of C.saxicola Bunting and pretreated sample for HSCCC. (A):n-butanol extract, (B): pretreated sample for furtherseparation on HSCCC; (C) peak B1 in Fig. 2B; (D) peakB2 in Fig. 2B; (E) peak B3 in Fig. 2B. Conditions: column,Phenomenex Gemini C18, 250 mm*4.6 mm ID, 5 μm;mobile phase, acetonitrile (A) and 20 mmol/L ammoniumacetate (B, adjusted to pH 4.66 by acetic acid), which wasprogrammed as: 0-10 min, A: 15-20%; 10-20 min, A:20-22%; 20-25 min, A: 22%; 25-30 min, A: 22-24%; 30-45min, 24-29%; 45-75 min, 29-90%; Flow rate, 1.0 mL/min;detection, 280 nm.

Figure 2. (A) HSCCC separation of pretreated sample ofn-BuOH extract of C.saxicola Bunting using solvent system:n-Hex-EA-n-BuOH-EtOH-W-buffer (pH=9) at 0.3：1.5：4：0.3：5：0.11 (v/v/v/v). Stationary phase, lower phase; coil column,231 mL; rotated speed, 860 rpm; flow rate, 1.5 mL/min;sample injection, 27.3 mg; detection wavelength, 280 nm; theratio retention of stationary phase was 81.4%.(B) HSCCC separation of the eluent of peak 1 in Fig.2A using

solvent system: n-Hex-EA-n-BuOH-EtOH-W-ammonia at 0.3：1.5：4：0.3：5：0.11 (v/v/v/v). Stationary phase, lower phase;coil volumn, 230 mL; rotated speed, 860 rpm; flow rate, 1.5mL/min; sample injection, 30.0 mg; detection wavelength, 280nm; the ratio retention of stationary phase was 82.7%.

ReferencesReferencesReferencesReferences1. Chemistry & Biodiversity, 2008, 5, 777-783.

2. Chemistry & Biodiversity, 2008, 5, 1335-1344.

mailto:Tel:+86 (020)39943042



OOOO-22-22-22-22SSSSeparationeparationeparationeparation andandandand purificationpurificationpurificationpurification ofofofof fourfourfourfour ligansligansligansligans fromfromfromfrom

fructusfructusfructusfructus arctiiarctiiarctiiarctii bybybyby high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyMeiMeiMeiMei YangYangYangYang,,,, XinXinXinXinjjjjunununun Xu*Xu*Xu*Xu*,,,, ZhiZhiZhiZhisssshenghenghengheng XieXieXieXie,,,, JieyunJieyunJieyunJieyun Huang,Huang,Huang,Huang, ChunChunChunChunyyyyanananan XieXieXieXie

School of Pharmaceutical Sciences, Sun Yat-Sen University* No. 132, East Waihuan Rd.,Guangzhou Higher Education Mega Center, 510006 Guangzhou, Chinae-mail address: [email protected]. fax: +86 020 39943041.

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

Four lignans including arctiin, arctigenin, matairesinol and lappaol F were separated and purified from traditionalChinese medicine Fructus Arctii by high-speed counter-current chromatogra--phy (HSCCC). The crude extracts fromFructus Arctii were divided into two fractions by D101 macroporous resin. Fraction 1 was separated with ethylacetate-n-butanol-water (4:0.5:5, v/v) and yielded 163.8 mg of arctiin from 250 mg of fraction 1. Fraction 2 wasseparated with n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v) and yielded 27 mg of arctigenin, 5 mg ofmatairesinol 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 compoundswere identified by MS, UV, 1H NMR and 13C NMR. Such a simple and effective method was fairly useful to preparepure compounds as reference substances for related study on Fructus Arctii.

FigureFigureFigureFigure 1111 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.



OOOO----22223333SeparationSeparationSeparationSeparation andandandand EnrichmentEnrichmentEnrichmentEnrichment ofofofof RareRareRareRare EarthEarthEarthEarth ElementsElementsElementsElements bybybyby StepwiseStepwiseStepwiseStepwise pHpHpHpH GradientGradientGradientGradientCountercurrentCountercurrentCountercurrentCountercurrent ChromatographyChromatographyChromatographyChromatography withwithwithwith aaaa PolyethylenePolyethylenePolyethylenePolyethylene Glycol-NaGlycol-NaGlycol-NaGlycol-Na2222SOSOSOSO4444 AqueousAqueousAqueousAqueous

Two-PhaseTwo-PhaseTwo-PhaseTwo-Phase SystemSystemSystemSystemMasamiMasamiMasamiMasami Shibukawa,*Shibukawa,*Shibukawa,*Shibukawa,* KoheiKoheiKoheiKohei Shimizu,Shimizu,Shimizu,Shimizu, ShingoShingoShingoShingo SaitoSaitoSaitoSaitoGraduate 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:Keywords:Keywords:Keywords: stepwise pH gradient CCC, pH-peak-focusing, rare earth elements, PEG-Na2SO4 aqueous two-phasesystem, acetylacetone.

In recent years it has been shown that high-speed countercurrent chromatography (HSCCC) can be applied forseparation of various metal ions. Kitazume et al.1, 2 demonstrated that HSCCC can also be used for efficientenrichment of rare earth elements and some other metal ions using pH-peak-focusing technique. However, most ofthe separations of metal ions by HSCCC which have so far been reported were performed with organic solvent-waterextraction systems. On the other hand, aqueous two-phase systems (ATPS) consisting of water-soluble polymers andinorganic salts have been known as an environmentally friendly extraction system and are used for separation ofinorganic ions as well as organic substances.3 In this paper, we will present an HSCCC method for separation of rareearth metal ions with a polyethylene glycol-Na2SO4 ATPS. We will also show that stepwise pH gradient technique isuseful 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-speedcounter-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 asthe stationary phase, while the salt-rich lower phase was used as the mobile phase. The column was first entirelyfilled with the stationary phase, and then the sample solution was injected through the sample port and the mobilephases 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 bymonitoring the absorbance at 650 nm with a spectrophotometer.

Rare earth metal ions were extracted into the PEG-rich phase by complexation with acetylacetone. Stabilityconstants of the complexes depend on the nature of the rare earth metal ions. Therefore, pH values at which theextraction takes place are also dependent on the type of the metal ions. This means that mutual separation of rareearth elements can be performed by HSCCC using a stepwise pH gradient elution. Furthermore enrichment of theindividual 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. Itshould be noted that not only mutual separation but enrichment of the metal ions are achieved. We will also discussthe mechanism of formation of pH gradient profile in the ATPS-HSCCC and the potential of this technique forpreparative scale separation.

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

Fig.1 Chromatogram of rare earth elements and pH profile of eluate.

Abso

rban

ce pH

0 50 100 150 200 time (min)

La Ce

Nd

Yb

Sc

pH 7.5 pH 7.1 pH 6.5 pH 5.9 pH 3.0HEPES BES PIPES MES H2SO4

Figure 1. Chromatogram of selected rare earth elementsobtained by stepwise pH gradient HSCCC

eferenceseferenceseferenceseferences1. Kitazume, E.; Higashiyama, T.; Sato, N.; Kanetomo, M.; Tajima, T.; Kobayashi, S.; Ito, Y. Anal. Chem. 1999199919991999, 71, 5515-5521.2. Kitazume, E.; Takatsuka, T.; Ito, Y. J. Liq. Chromatogr. & Rel. Technol. 2004200420042004, 27, 437-449.3. Shibukawa, M.; Nakayama, N.; Hayashi, T.; Shibuya, D.; Endo, Y.; Kawamura, S. Anal. Chim. Acta 2001200120012001, 427, 293-300.


OOOO----22224444ApplicationApplicationApplicationApplication ofofofof High-speedHigh-speedHigh-speedHigh-speed CountercurrentCountercurrentCountercurrentCountercurrent Chromatography,Chromatography,Chromatography,Chromatography, TLCTLCTLCTLC andandandand

nano-nano-nano-nano-LC-MS/MSLC-MS/MSLC-MS/MSLC-MS/MS inininin LipidomicsLipidomicsLipidomicsLipidomics

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 speciesexisting in the cells and tissues. Lipids play multiple and critical roles in cellular functions, suchas composing the membrane bilayer, providing an appropriate hydrophobic environment formembrane proteins and their interactions, and participating in cell growth, multiplication, anddeath. Simultaneously, some lipids are messengers in cell signaling transduction processes andcan 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 livingsystem. Comparing the lipidome of healthy versus diseased states can provide informationhelpful in correlating the role of lipids in various diseases, such as cancer, atherosclerosis, andchronic inflammation. Additionally, a large variety of lipid species comprise cellular membranes,and investigating their interactions with membrane-associating proteins/enzymes can provideinsight 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 ofhydrophobicity. As a result of the complexity and diversity of lipids, efficient separation andaccurate 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 andopen 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 andCaenorhabditis elegans, followed by TLC and HPLC-ESI-MS/MS analysis. Four two-phasesolvent systems are tested for HSCCC separation in this study.


OOOO----22225555IIIIsolationsolationsolationsolation ofofofof twotwotwotwo newnewnewnew diterpenoidsditerpenoidsditerpenoidsditerpenoids fromfromfromfrom clerodendrumclerodendrumclerodendrumclerodendrum kaichianumkaichianumkaichianumkaichianum hsu.hsu.hsu.hsu. bybybyby

high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography usingusingusingusing stepwisestepwisestepwisestepwise elutionelutionelutionelutionMing-fengMing-fengMing-fengMing-feng XuXuXuXuaaaa,,,, Qi-zhenQi-zhenQi-zhenQi-zhen DuDuDuDub,*b,*b,*b,*,,,, Lian-qingLian-qingLian-qingLian-qing ShenShenShenShenbbbb,,,, Hui-zhongHui-zhongHui-zhongHui-zhong WangWangWangWangaaaa

aCollege of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, ChinabCollege 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:Keywords:Keywords: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 andwarm temperate zones including Africa and southern Asia. A few of species are distributed in America, northernAustralia 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 chromatographyusing stepwise elution was studied in this paper. Since the different retention of stationary phase of different systemcomposition, the change of speed was studied in order to maintain the retention of stationary phase with differentsystem. After the flow rate of the HSCCC system was adjusted to give the same retention of stationary phase as thatof the first system, these optimized parameters were successfully change to the second system for the separation andpurification of four compounds from C. Kaichianum.

The HSCCC was performed on a HSCCC instrument equipped with a 1100-mL column, using the upper phase oflight petroleum–ethyl acetate–methanol–water (PEMW) (5:5:3:6, v/v/v/v) as stationary phase, and the lower phase ofPEMW as mobile phase, later changed to (PEMW) (5:5:5:5). Using the optimized conditions, two new and twoknown diterpenoids were obstained, 2 g of crude extract was separated to yield 19.3 mg of A, 25.9 mg of B, 12.4 mgof C and 18.6 mg of D with purities 81.2%, 85.7%, 95.3% and 96.2%, respectively. Their structures were identifiedby 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 isolatedand purified by the stepwise counter-current chromatography method, which demonstrated that stepwise HSCCC is afast, 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) 1994199419941994. 65, pp. 150-151.2. H.H. Nan, J. Wu, and S. Zhang, Pharmazie 2005200520052005,60, 798.3. S.S. Liu, T.Z. Zhou, S.W. Zhang, and L.J. Xuan, Helv. Chim. Acta 2009200920092009, 92, 1070.4. R. Ravikumar, A.J. Lakshmanan, and S. Ravi, J. Asian Nat. Prod. Res. 2008200820082008, 10, 652.5. T. Kanchanapoom, P. Chumsri, R. Kasai, H. Otsuka, and K. Yamasaki, J. Asian Nat. Prod. Res. 2008200820082008, 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.

http://en.wikipedia.org/wiki/Verbenaceae

http://en.wikipedia.org/wiki/Africa

http://en.wikipedia.org/wiki/Asia

http://en.wikipedia.org/wiki/Americas


OOOO-26-26-26-26PreparativePreparativePreparativePreparative isolationisolationisolationisolation ofofofof OolongOolongOolongOolong teateateatea polyphenolspolyphenolspolyphenolspolyphenols bybybyby high-speedhigh-speedhigh-speedhigh-speed countercurrentcountercurrentcountercurrentcountercurrent

chromatographychromatographychromatographychromatographyKunboKunboKunboKunbo Wang,Wang,Wang,Wang, FangFangFangFang Liu,Liu,Liu,Liu, ZhonghuaZhonghuaZhonghuaZhonghua Liu*,Liu*,Liu*,Liu*, Jian-anJian-anJian-anJian-an Huang,Huang,Huang,Huang, YongYongYongYong Lin,Lin,Lin,Lin, YushunYushunYushunYushun GongGongGongGong

National Research Center of Engineering & Technology for Utilization of Botanical Functional Ingredients, KeyLaboratory 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 Republicof China, email: [email protected]

Keywords:Keywords:Keywords:Keywords:High-speed countercurrent chromatography; Oolong tea; catechins.

Abstract: The characteristic feature of oolong tea components is that it contains numerous kinds ofpolymerized-polyphenols, which are derived from tea catechins by polyphenol oxidases or by heating process, whichis 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 hasbeen based on chromatography on (semi-) preparative HPLC and sephadex LH-20 (2,3). The procedures are timesconsuming 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-ethylacetate-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. Theresults indicated that catechins including epigallocatechin gallate, gallocatechin gallate and epicatechin gallate wereisolated from the aqueous extract of oolong tea (Fig.1). Fujian Tieguanyin, Dahongpao, Guangdong FenghuangDancong 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 aqueousphase; 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.


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) 34255. 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.



OOOO-27-27-27-27GenerationGenerationGenerationGeneration ofofofof knock-outknock-outknock-outknock-out extractsextractsextractsextracts bybybyby countercurrentcountercurrentcountercurrentcountercurrent chemicalchemicalchemicalchemical subtractionsubtractionsubtractionsubtraction

RenRenRenRenéééé F.F.F.F. Ramos,Ramos,Ramos,Ramos, GuidoGuidoGuidoGuido F.F.F.F. Pauli,Pauli,Pauli,Pauli, andandandand Shao-NongShao-NongShao-NongShao-Nong ChenChenChenChen1,1,1,1,****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:Keywords:Keywords: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 anyseparation technology. In particular, solid-phase based chromatography suffers from unavoidable absorption, makingit essentially unsuitable for studies involving bioactivity and quantitation. Suitable methods for chemical subtractionare 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 withmonoclonal antibodies for the removal of single phytoconstituents from plant extracts. Another approach, termedchemical subtraction, has been pioneered in our laboratory and uses countercurrent chromatography (CCC) todeplete 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 ofboth 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 bioactiveprenylated phenols contained in the dietary supplement, hops: xanthohumol [XH], isoxanthohumol [IX], and8-prenylnaringenin [8-PN]. The approach uses HSCCC and HEMWat 0 as the initial solvent system. Reflecting theirdifferent 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-elutionwith 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 targetedanalysis, 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.


OOOO-28-28-28-28RapidRapidRapidRapid SeparationSeparationSeparationSeparation ofofofof PolyphenolsPolyphenolsPolyphenolsPolyphenols fromfromfromfrom GreenGreenGreenGreen TeaTeaTeaTea bybybyby HSCCCHSCCCHSCCCHSCCC andandandand

CharacterizationCharacterizationCharacterizationCharacterization thethethethe interactioninteractioninteractioninteraction betweenbetweenbetweenbetween EGCGEGCGEGCGEGCG andandandand ProteinProteinProteinProtein bybybyby ACEACEACEACEWANGWANGWANGWANG Wei*,Wei*,Wei*,Wei*, LELELELE Sheng-feng,Sheng-feng,Sheng-feng,Sheng-feng, ZHAOZHAOZHAOZHAO Xin-ying,Xin-ying,Xin-ying,Xin-ying, WANGWANGWANGWANG Tan,Tan,Tan,Tan, ZHANGZHANGZHANGZHANG Jing-huaJing-huaJing-huaJing-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:Keywords:Keywords: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, suchas catechins, flavonoid glycosides and phenolic acids. Among of them epigallocatechin gallate (EGCG) is regardedas the most important of the tea catechins. It has been recognized that the catechins possess many biologicalactivities and pharmacological actions including antioxidant, anti-allergy, anti-virus, reduced risk of cardio-vascularinjury et al.

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

The interaction between EGCG and protein (Bovine Serum Albumin, BSA) was determined by affinity capillaryelectrophoresis (ACE). The separation was performed at 40/48.5 cm capillary with 10 kV voltage and 214 nmdetection. The electrophoretic mobility changes of EGCG were measured with various concentrations of BSA addedto the borax running buffer, pH 8.7 (Figure 2). The binding constant was calculated from the electrophoretic mobilitychange 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 constantbetween EGCG and BSA determined by ACE is in good agreement with that reported in the literature. The presentwork offers a good quick method for study on affinity interactions of drug-protein from crude extract, which can begenerally applied to a wide of screening active ingredients.


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.

Figure 1 Chromatograms of crude polyphenolextract from green tea obtained by HSCCC

Figure 2 Electropherograms of EGCG andBSA based on ACE1 mM EGCG and BSA 0 μM (A). BSA 3 μM(B). BSA 5 μM. (C) BSA 10 μM. (D) BSA 15μM (E).


OOOO-29-29-29-29OnlineOnlineOnlineOnline isolationisolationisolationisolation andandandand purificationpurificationpurificationpurification ofofofof fourfourfourfour phthalidephthalidephthalidephthalide compoundscompoundscompoundscompounds fromfromfromfrom RRRRhizomehizomehizomehizome

chuanxiongchuanxiongchuanxiongchuanxiong usingusingusingusing counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography coupledcoupledcoupledcoupled withwithwithwith semisemisemisemi preparativepreparativepreparativepreparativehighhighhighhigh performanceperformanceperformanceperformance liquidliquidliquidliquid chromatographychromatographychromatographychromatography

YunYunYunYun WeiWeiWeiWei ****,,,, WenwenWenwenWenwenWenwen HuangHuangHuangHuang*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:Keywords:Keywords:Keywords: Phthalide Compounds, Counter-current Chromatography, Semi Preparative High Performance LiquidChromatography

ABSTRACTABSTRACTABSTRACTABSTRACTRhizome chuanxiong is one of the most widely used traditional herbal medicines. There are three types ofpharmacologically 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) and3-butylidenephthalide (4), have been reported as the biologically active compounds contributing to the therapeuticeffects of this medicinal herb[1]. In this study, a counter-current chromatography coupled with semi preparative highperformance liquid chromatography through a six-way valve has been used to achieve online isolation andpurification 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 chromatographyseparation [2-3]. So a six-way valve was used to cut the peak of the mixture and injected to the semi preparative highperformance liquid chromatography directly. By using a solvent system ofn-hexane-ethylacetate-methanol-water-acetonitrile ( 8:2:5:5:5, v/v ) , 3.6 mg (1) (94.43%), 3.0 mg (2) (95.31%) wereobtained, and the 8 ml of (3)+(4) were transferred and injected for further separation by the semi preparative highperformance liquid chromatography. 5.6 mg (3) (97.5%) and 4.8 mg (4) (99.3%) were obtained. The identification ofthe four compounds were performed by ESI-MS, 1H-NMR and 13C-NMR spectra.

ReferencesReferencesReferencesReferences(1) Yan, R.; Li, S.L.; Chung, H.S.; Tam, Y.K.; Lin, G. J. Pharm. Biomed. Anal.2005200520052005, 37, 87-95

(2) Wei, Y.; Xie, Q.Q.; Dong, W.T.; Ito, Y. J. Chromatogr. A. 2009200920092009,1216, 4313–4318.(3) Beer, D.; Jerz, G.; Joubert, E.; Wray, V.; Winterhalter, P. J. Chromatogr. A. 2009200920092009,1216, 4282–4289.

0 5 10 15 20 25 30 35 40 45 50

0

10

20

30

40

50

60

70

80

90

100

110

mA

u

time(min)

3

4

Figure 1 Scheme of the CCC-Pre-HPLC systemFigure 2. Chromatogram of the crude extract fromRhizome chuanxiong by HSCCC. solventsystem :n-hexane-ethylacetate-methanol-water-acetonitrile ( 8:2:5:5:5, v/v ); sample: 100 mg crudeextract dissolved in 0.5 ml lower phase and 0.5 mlupper phase; 1 = senkyunolide A; 2 = levistolide A;3=Z-ligustilide; 4=3-butylidenephthalide .

Figure 3. Chromatogram of mixture of (3)+(4) by Pre-HPLC. reversed phaseC18 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.


OOOO----33330000SeparationSeparationSeparationSeparation ofofofof 11-cis-retinal11-cis-retinal11-cis-retinal11-cis-retinal fromfromfromfrom thethethethe retinalretinalretinalretinal isomersisomersisomersisomers bybybyby flashflashflashflash countercurrentcountercurrentcountercurrentcountercurrent

chromatographychromatographychromatographychromatography1111MinfeiMinfeiMinfeiMinfei He,He,He,He, 1111WenkaiWenkaiWenkaiWenkai Du,Du,Du,Du, 1111QingbaoQingbaoQingbaoQingbao Du,Du,Du,Du, 1111YunYunYunYun Zhang,Zhang,Zhang,Zhang, 1111BoBoBoBo Li,Li,Li,Li, 2222ChangqianChangqianChangqianChangqian Ke,Ke,Ke,Ke, 2222YangYangYangYang

Ye*,Ye*,Ye*,Ye*, 1111QizhenQizhenQizhenQizhen Du*Du*Du*Du*1Institute of Food Chemistry, Zhejiang Gongshang University, Hangzhou 3100125, China

2State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academyof 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:Keywords:Keywords: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. Theinstability of 11-cis-retinal causes lack of commercial supply. The preparation of 11-cis-retinal has been achieving byan isomerization reaction of all-trans-retinal. But the separation of 11-cis-retinal from the reaction products was soeasy, 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 limitedamount can be applied to a plate, (ii) the separation of the 11-cis and 13-cis isomers is poor, (iii) the material isparticularly prone to degradation while adsorbed on the stationary phase. Following, high-performance liquidchromato-graphy (HPLC) has been applying to the separation of retinal isomers, but the loading amount was lowerthan 1 mg of the products of the isomerization reaction on a preparative column (Supelcosil LC-Si, 250 x 21.2 mm, 5µm). In the present paper, we describe a HSCCC separation of the products of the isomerization reaction with amount of sample load more than 60 milligram.

O 9

11

13

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-pressuremercury arc lamp (Thorn Lighting, Type MED) with another as a stopper. The light is passed through aheat-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 reactionsolution was cooled to 3°C and an irradiation was performed for 2 min. After the irradiation finished, the solution ofthe 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 tosaturation of n-hexane (40 ml). The mobile phase was pumped at a flow rate of 25.0 ml/min when the apparatus wasrotated at 1000 rpm. The effluent was monitored by a UV detector at 236 nm and collected by a fraction collectorwith 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). Columncapacity: 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 of9-cis-retinal with a purity of 97%. The method can meet the preparation for the research of bioactivity of11-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.



OOOO----33331111Nmr-guidedNmr-guidedNmr-guidedNmr-guided countercurrentcountercurrentcountercurrentcountercurrent purificationpurificationpurificationpurification andandandand quantificationquantificationquantificationquantification ofofofof greengreengreengreen teateateatea catechinscatechinscatechinscatechins

FengFengFengFeng QiuQiuQiuQiu1111,,,, SamuelSamuelSamuelSamuel ProProProPro2222,,,, GuidoGuidoGuidoGuido F.F.F.F. PauliPauliPauliPauli1111,,,, J.J.J.J. BrentBrentBrentBrent FriesenFriesenFriesenFriesen1,3,1,3,1,3,1,3,****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:Keywords:Keywords:Keywords: qNMR, Camellia sinensis, catechins, countercurrent separation

Nuclear Magnetic Resonance (NMR) was used to measure K values, identify analytes, and determine product purityfor the high-speed countercurrent chromatography (HSCCC) isolation of major and minor catechins from anethanolic extract of green tea (Camellia sinensis). Four possible solvent systems (SSs) for the separation of green teacatechins were identified by the Generally Useful Estimation of Solvent Systems (GUESS) method. The K values ofcatechin (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-flaskexperiment. At the same time, the presence of other phytoconstituents such as caffeine and gallocatechin gallatecould also be observed in the NMR spectra. Three rounds of NMR-guided SS selection were performed to chooseappropriate 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 withorthogonal characteristics. Analytical and preparative scale HSCCC separations were performed on a prototypeCherryOne 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 analysisof the separation process. Each CCC fraction was weighed in order to create a mass chromatogram of the entireseparation. NMR of analysis of combined fractions from the primary fractionation step facilitated the identificationof analytes and informed the selection of the appropriate secondary fraction steps employing orthogonal SS in whichpurified green tea catechins were obtained and measured for purity with qHNMR techniques. NMR-guided isolationand gravimetric quantification of target analytes avoids several challenges associated with HPLC-guided isolationand analysis such as: (a) the need to develop a chromatographic method for HPLC; (b) acquisition andcharacterization of identical reference standards required to identify and quantitate HPLC peaks; (c) variation ofsensitivity inherent in different detection methods that influences quantitation; and (d) the transparency of certainimpurities in HPLC detection.

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


OOOO----33332222SeparationSeparationSeparationSeparation ofofofof bioactivebioactivebioactivebioactive componentscomponentscomponentscomponents fromfromfromfrom GynuraGynuraGynuraGynura divaricatadivaricatadivaricatadivaricata(L.)(L.)(L.)(L.) DCDCDCDC bybybyby high-speedhigh-speedhigh-speedhigh-speed

counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyLuLuLuLu Yin,Yin,Yin,Yin, XueliXueliXueliXueli Cao*,Cao*,Cao*,Cao*, JingJingJingJing Xu,Xu,Xu,Xu, ChaoChaoChaoChao Cheng,Cheng,Cheng,Cheng, HairunHairunHairunHairun PeiPeiPeiPei

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:Keywords:Keywords:Keywords: Gynura divaricata(L.) DC.; High-speed countercurrent chromatography (HSCCC); bioactivecomponents

Gynura divaricata (L.) DC is a traditional medicinal andedible plant in China. With its authentication as a newresources food by China State food & Drug Administrationin 2010, a variety of its biological effects and healthfunctions draw much more public attention, especially itshypoglycemic effect. However, the material basis of thiseffect is not very clear at this moment, more intensive andsystematic research is necessary. In order to investigatepotential bioactive components from Gynura divaricata(L.)DC., the 65% ethanol extract of the stem and leaf ofGynura divaricata(L.) DC. was fractioned by extractionwith petroleum ether, ethyl acetate and n-butanolsuccessively. According to the results of active assay, theethyl acetate fraction was subjected to further separation.In this paper, four phenolic acids and one flavonoid wereseparated and purified from Gynura divaricata(L.) DC.using high-speed counter-current chromatography(HSCCC). The solvent system was selected by analyticHSCCC 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 employedfor preparative separation. Four pure compounds wereobtained within 60min from 300 mg ethyl acetate extract,including 8.8 mg of 4,5-dicaffeoylquinic acid (Isochlorogenic acid C, 2222), 35.1 mg of 3,4-dicaffeoylquinic acid(Isochlorogenic acid B, 3333), 12.6 mg of 3,5-dicaffeoylquinic acid (Isochlorogenic acid A, 4444) and 10.6 mg ofkaempferol-3-O-β-D-glucopyranoside (Astragalin, 5555). Then, 22.4 mg of 3-O-caffeoylquinic acid(Chlorogenic acid,1111) 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% determinedby HPLC. The chemical structures were confirmed by 1H-NMR, 13C-NMR and ESI-MS.


1. Chunpeng Wan, Yanying Yu, Shouran Zhou, Shuge Tian, and Shuwen Cao. Isolation and identification of phenolic compounds from Gynuradivaricata 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

Figure 1. The structures of compounds purified from

Gynura divaricata(L.) DC.



OOOO----33333333CombinationCombinationCombinationCombination ofofofof High-SpeedHigh-SpeedHigh-SpeedHigh-Speed Counter-CurrentCounter-CurrentCounter-CurrentCounter-Current ChromatographyChromatographyChromatographyChromatography andandandand PreparativePreparativePreparativePreparativeHPLCHPLCHPLCHPLC totototo SeparationSeparationSeparationSeparation ofofofof FlavonoidFlavonoidFlavonoidFlavonoid GlycosidesGlycosidesGlycosidesGlycosides andandandand CaffeoylquinicCaffeoylquinicCaffeoylquinicCaffeoylquinic AcidAcidAcidAcid DerivativesDerivativesDerivativesDerivatives

fromfromfromfrom LeavesLeavesLeavesLeaves ofofofof LoniceraLoniceraLoniceraLonicera japonicajaponicajaponicajaponicaThunb.Thunb.Thunb.Thunb.DaijieDaijieDaijieDaijie WangWangWangWang1,1,1,1, 2222,,,, JinhuaJinhuaJinhuaJinhua DuDuDuDu1111*,*,*,*, YunLiangYunLiangYunLiangYunLiang LinLinLinLin2222,,,, ShengboShengboShengboShengbo LiLiLiLi3333,,,, XiaoXiaoXiaoXiao WangWangWangWang2222

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:Keywords:Keywords:Keywords: Lonicera japonica Thunb.; flavonoid glycosides; caffeoylquinic acid derivatives; high-speedcounter-current chromatography; preparative HPLC

ABSTRACTABSTRACTABSTRACTABSTRACT Lonicera japonica Thunb. (Caprifoliaceae plants) is one of the most common traditional Chinesemedicines and used for treating various diseases, including arthritis, diabetes mellitus, fever and infections. Theleaves of L. Japonica is not fully research, it is necessary to develope an efficient method to separate and purifyactive 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 caffeoylquinicacid derivatives. 1.0 g of the n-butanol extract was firstly isolated by HSCCC using a two-phase solvent systemcomposed of methyl tert-butyl ether-n-butanol-acetonitrile-water (0.5% acetic acid) (2: 2: 1: 5, v/v), yielding fivefractions 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 andluteoloside. F4 (mixed with 3,4-O-dicaffeoylquinic acid and hyperoside) and F5 (mixed with 3,5-O-dicaffeoylquinicacid and 4,5-O-dicaffeoylquinic acid) were separated by preparatve HPLC successfully. Combination these twomethods, 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 of3,5-O-dicaffeoylquinic acid (8) and 26.1 mg of 4,5-O-dicaffeoylquinic acid (9) were yielded, with the purities of99.5%, 98.7%, 99.3%, 94.3%, 96.1%, 97.1%, 97.4%, 96.9% and 97.8%, respectively, as determined by HPLC. Thestructures 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 ofsample on the solid support. It is an excellent enrich method for separation similar polity compounds.

Fig. 1. The HPLC analysis of the n-butanol extractFig. 2. HSCCC chromatogram of n-butanol extract

Fig. 3. The structures of isolated nine compounds

µ µ µ µ µ µ µ µ µµµt /h

µµA

bsor

banc

e(2

54nm

)

µµF

1 µµF

2

µµF

3µµF

4

µµt/min

µµ

µ

µ

µ

µµ

µ

µ

R2O

OR1 OH

R3O

CO2H123

45 6

HO

COO

Caffeoyl=

HO

1'

2'3'

4'

5' 6'

7'

8' 9'8 R1=caffeoyl R2=H R3=caffeoyl

6 R1=caffeoyl R2=caffeoyl R3=H

9 R1=H R2=caffeoyl R3=caffeoyl

1 R1=caffeoyl R2=H R3=H

O

OOH

R2 R3

OH

R1

2 R1=H R2=OGlc(2-1)Rha R3=OH

3 R1=OGlc(6-1)Rha R2=OH R3=OH

4 R1=H R2=OGlc(2-1)Rha R3=H

5 R1=H R2=OGlc R3=OH

7 R1=OGal R2=OH R3=OH



OOOO----33334444PPPPreparativereparativereparativereparative separationseparationseparationseparation ofofofof conjugatedconjugatedconjugatedconjugated linoleiclinoleiclinoleiclinoleic acidsacidsacidsacids (CLA)(CLA)(CLA)(CLA) fromfromfromfrom camelliacamelliacamelliacamellia oleiferaoleiferaoleiferaoleifera

abelabelabelabel usingusingusingusing ph-zone-refiningph-zone-refiningph-zone-refiningph-zone-refining counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyGuangleiGuangleiGuangleiGuanglei Song,Song,Song,Song, JingboJingboJingboJingbo Wang,Wang,Wang,Wang, YanboYanboYanboYanbo Wang,Wang,Wang,Wang, QizhenQizhenQizhenQizhen Du*Du*Du*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:Keywords:Keywords:Keywords: Camellia oleifera Abel, Conjugated linoleic acid, pH-zone-refining high-speed counter-currentchromatography, 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 and13-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. Theseparation (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-octadecadienoateand 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).


1. Pajunen, T. I., Koskela, H., Hase, T., Hopia, A. Chem. Phys. Lipids. 2008, 154, 1052. Tong, S., Yan, J., Guan, Y. X. J. Chromatogr. A 2008,1212, 483. Weisz, A., Idina, A., Ben-Ari, J., Karni, M., Mandelbaum, A., Ito, Y. J. Chromatogr. A 2007, 1151, 824. 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


OOOO----33335555FromFromFromFrom medicinalmedicinalmedicinalmedicinal plantplantplantplant totototo compound:compound:compound:compound: isolationisolationisolationisolation ofofofof shikimicshikimicshikimicshikimic acidacidacidacid fromfromfromfrom IlliciumIlliciumIlliciumIllicium verumverumverumverum

bybybyby hyphenatedhyphenatedhyphenatedhyphenated expandedexpandedexpandedexpanded bedbedbedbed adsorptionadsorptionadsorptionadsorption chromatographychromatographychromatographychromatography andandandand counter-currentcounter-currentcounter-currentcounter-currentchromatographychromatographychromatographychromatography

PingPingPingPing HuHuHuHu1111,,,, ManManManMan LiuLiuLiuLiu1111,,,, MinMinMinMin ZhangZhangZhangZhang2,2,2,2, ****,,,, YingxiYingxiYingxiYingxi HuaHuaHuaHua1111,,,,HongyangHongyangHongyangHongyang ZhangZhangZhangZhang1111,,,, YuerongYuerongYuerongYuerong WangWangWangWang1111,,,, GuoanGuoanGuoanGuoan LuoLuoLuoLuo2,32,32,32,3 ********

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

AbstractAbstractAbstractAbstract

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 shortprocessing time [1]. Natural compounds, which researchers are interested in, are usually present in medicinal plants atvery 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 preliminaryseparation 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 adsorptionchromatography (EBAC) is a unique technique which integrated clarification, concentration and purification into oneoperation 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 methodinvolves: 1) hyphenating EBAC to CCC via an interface unit, 2) in the 1st dimension, preliminary separation oftargets by integrated EBAC, 3) CCC solvent system selection and matching, 4) in the 2nd dimension, fine separationof targets by CCC.

This method was used in the following case study. In the extraction step, a certain amount (8.0 g) of Illicium verumwas put into the continuous extraction column with an ultrasonic assisted extraction device outside. Ion exchangeresins (D293) were packed into the expanded bed chromatography column. Target compound-shikimic acid wasextracted in the continuous extraction column and simultaneously the target being extracted was directly exchangedonto the resins packed in the expanded bed column. During this operation, the resins in chromatographic columnwere in an expanded status and there was space among resins. This character of expanded bed will allow particulatecontaining feed stock to go through the column without blockage. In the 1st dimension, the expanded bed was elutedby 2% aqueous NaCl solution and part of the fractions with shikimic acid was transferred to the interface unit-asample 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 wasperformed to separate shikimic acid with a butanol-water-acetic acid (4:5:1) solvent system. 28-mg of shikimic acidwas purified, with 99.6% purity and 50% recovery. This research case study proved the feasibility of the newlydeveloped hyphenated chromatography method.

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

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


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


OOOO-36-36-36-36ComprehensiveComprehensiveComprehensiveComprehensive SeparationSeparationSeparationSeparation ofofofof LignansLignansLignansLignans fromfromfromfrom ZanthoxylumZanthoxylumZanthoxylumZanthoxylum planispinumplanispinumplanispinumplanispinum bybybyby

Counter-CurrentCounter-CurrentCounter-CurrentCounter-Current ChromatographyChromatographyChromatographyChromatography CombinedCombinedCombinedCombined withwithwithwith High-PerformanceHigh-PerformanceHigh-PerformanceHigh-Performance LiquidLiquidLiquidLiquidChromatography/TandemChromatography/TandemChromatography/TandemChromatography/Tandem MassMassMassMass SpectrometrySpectrometrySpectrometrySpectrometry

YanbinYanbinYanbinYanbin Lu*,Lu*,Lu*,Lu*, XianjiangXianjiangXianjiangXianjiang Lin,Lin,Lin,Lin, KuiwuKuiwuKuiwuKuiwu Wang,Wang,Wang,Wang,College of Food and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China

*fax +86-571-88071024-7587, e-mail: [email protected]

Keywords:eywords:eywords:eywords: Counter-current chromatography; Comprehensive Separation; High-performance LiquidChromatography/Tandem Mass Spectrometry; Zanthoxylum planispinum; Lignans.

An effective method combing fast counter-current chromatography (CCC) and HPLC/MS for comprehensiveseparation 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 thegolden 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 presentwork, 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. Asa 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.Fig.Fig.Fig. 1111 HPLC analysis of the crude extract.

Fig.Fig.Fig.Fig. 2222 CCC separation of the target compounds with different

conditions.


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.


OOOO-37-37-37-37LLLLarge-scalearge-scalearge-scalearge-scale separationseparationseparationseparation ofofofof alkaloidsalkaloidsalkaloidsalkaloids fromfromfromfrom CCCCorydalisorydalisorydalisorydalis bungeanabungeanabungeanabungeana turca.turca.turca.turca. bybybyby

pHpHpHpH----ZZZZone-one-one-one-FFFFefiningefiningefiningefining counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyXiaoXiaoXiaoXiao WangWangWangWang1111,,,, DonghongjingDonghongjingDonghongjingDonghongjing2222,Yangbin,Yangbin,Yangbin,Yangbin1111,,,, LinLinLinLin xiaojingxiaojingxiaojingxiaojing2222,Huangluqi,Huangluqi,Huangluqi,Huangluqi1111****

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, Shandong250014, China

*Correspondence: Huang Luqi, Institute of Chinese Medical Academy of Chinese Medical Science, 16Dongzhimennei Street, Beijing 100700, China. E-mail: [email protected] Fax: +86-10 -8402-7175

Keywords:Keywords:Keywords: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 asnorthern and eastern parts of China, the northern part of Korean peninsula, ect. (1). Its use for the treatment ofinfluenza, upper respiratory tract infections, bronchitis, tonsillitis, acute nephritis and pyelonephritis have beenreported 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 Corydalisbungeana 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 usedsuccessfully for the large-scale purification of a varieties of natural products (5). The aim of this paper was todevelop an efficient method for the large-scale separation of alkaloids from Corydalis bungeana Turcz. bypH-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) tothe 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.


[1] Chen, X., Nigel, C. V., Peter, J. H., Monique, S. S., Phytochemistry 2004200420042004, 65, 3041–3047.[2] Qingdao TCM Research Group, 1972197219721972. Zhong Cao Yao Tong Xun 5, 16.[3] Xie, C. Kokubun, T., Houghton, P. J. Simmonds, M. D. J., phytotherapy research 2004200420042004, 18:497-500.[4] Yang J. G., Yuan H. N.,, Che J., Zhang Q., Journal of clinical pharmacology 1990199019901990, 6, 35–36.[5] Ito, Y., J. Chromatogr. A 2005200520052005, 1065, 145–168.


http://dict.cnki.net/dict_result.aspx?searchword=%e8%8d%af%e7%90%86%e5%ad%a6%e7%9a%84&tjType=sentence&style=&t=pharmacologic


OOOO-38-38-38-38SeparationSeparationSeparationSeparation ofofofof thethethethe syntheticsyntheticsyntheticsynthetic productsproductsproductsproducts ofofofof acetylatedacetylatedacetylatedacetylated rheinrheinrheinrhein bybybyby high-speedhigh-speedhigh-speedhigh-speed

counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography

YindiYindiYindiYindi Zhu,Zhu,Zhu,Zhu, YingYingYingYing Zhan,Zhan,Zhan,Zhan, HaishuiHaishuiHaishuiHaishui Gui,Gui,Gui,Gui, QianQianQianQian Wei,Wei,Wei,Wei, YangyangYangyangYangyangYangyang Liu,Liu,Liu,Liu, TunhaiTunhaiTunhaiTunhai Xu*Xu*Xu*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:Keywords:Keywords: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 lowtoxicity and safety features, it has been concerned by many researchers [1]. Diacerein, the rhein acetylated syntheticproduct, which is a new type interleukin-21B (IL21B) inhibitor, can inhibit the activity of interleukin 21 (IL21) andIL2l synthesis in synovium [2]. For the similar structural among rhein acetylated synthetic products, especially theisomers of 1-acetylrhein and 8-acetylrhein, conventional chromatography is difficult to separate effectively, hinderthe further activity study. In this study, high-speed counter-current chromatography (hsccc) was applied to seperaterhein, 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 than98% analyzed by HPLC, respectively.

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

ReferencesReferencesReferencesReferences1. 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.


Authors Title Code

Eli, Y.; Yang, Y.; Yili, A.; Aisa, H.A.*

Preparative separation and purification of chemicalcompositions from Mentha longifolia L. by normal andreverse phase high-speed counter-current chromatography

PPPP-1-1-1-1

Fang, L.; Li, J.; Lin, Y.; Zhou, J.;Geng, Y.; Wang, X.*

Preparative separation and purification of five oxindolealkaloids from Gelsemium elegans by pH-zone-refiningcounter-current chromatography

PPPP-2-2-2-2

Faure, K.*; Mekaoui, N.; Berthod,A.

Evaluation of Limonene as a possible “green” Non polarsolvent for alkane replacement

PPPP-3-3-3-3

Gu, D.; Yang, Y.; Aisa, H. A., Ito,Y. *

Novel design for centrifugal counter-current chromatography:Ⅵ. Ellipsoid column

PPPP-4-4-4-4

Han, Q.A new two-phase solvent system of chloroform/ acetyl acetate/methanol/ water and its use in HSCCC separation ofaconitines from aconite roots

PPPP-5-5-5-5

Huang, J.; Xu, X.*; Xie, Z.; Yang,M.; Xie, C.

Isolation and purification of ecdysterone from Serratulachinensis S. Moore by high-speed counter currentchromatography

PPPP-6-6-6-6

Huang, X.; Chen, X.; Pei, D.;Zhang, J.; Di, D. *

Dual-mode counter-current chromatography applied toisolation maslinic and oleanolic acids from the olive millingsubproduct

PPPP-7-7-7-7

Kim, T. B.; Kim, H. W.; Sung, S.H.*

Preparative separation of major constituents in OPuntiaficus-indica by high-speed countercurrent chromatography

PPPP-8-8-8-8

Kitazume, E.*; Sudo, T.; Kubo, H.;Watanabe, M.; Ito, Y.

Elimination of metal elements by high-speed counter-currentchromatography by introducing air and solid absorbents

PPPP-9-9-9-9

Lee, K. J.; Ha, I. J.; Kim, Y. S. *Preparative isolation of chemical constituents from the roots ofPhlomis umbrosa by high-speed counter-currentchromatography

PPPP-10-10-10-10

Li, L.; Yang, Y.; Hou, X.; Ba, H.;Gu, D. ; Abdulla, R.; Xin, X.; Wu,G.; Aisa, H.A. *

Bioassay-guided separation and purification of water-solubleantioxidants from Carthamus tinctorius L. by combination ofchromatographic techniques

PPPP-11-11-11-11

Liang, J.; Meng, J.; Wu, S.*Preparative isolation and purification of tanshinones fromSalvia miltiorrhiza Bunge by a novel counter-currentchromatography with an upright conical coils

PPPP-12-12-12-12

Liang, Y.; Ma, Y.; Ito, Y. *Preparative separation of three flavonoids from the flowerbuds of Daphne genkwa by pH-zone-refining vortexcounter-current chromatography

PPPP-13-13-13-13


Authors Title Code

Mekaoui, N.*; Faure, K.; Berthod,A.

Multiple dual mode and trapping molecules for Coomassiebrilliant blue G-250 purification

PPPP-14-14-14-14

Meng, J.; Wu, S.*Multi-dimension counter-current chromatography for high-throughput analysis of natural products

PPPP-15-15-15-15

Shiono, H.*; Matsui, T.; Okada, T.;Chen, H.M.; Ito, Y.

Gentle and high-yield preparative separation method ofbasophils using a rotary-seal-free continuous-flow centrifuge

PPPP-16-16-16-16

Spórna-Kucab, A.*; Ignatova, S.;Garrard, I.; Wybraniec, S.

Betalain separation from red beet juice (Reta vularis L.) byhigh-performance countercurrent chromatography (HPCCC)in polar high-salt solvent systems

PPPP-17-17-17-17

Spórna-Kucab, A.*; Ignatova, S.;Garrard, I.; Wybraniec, S.

Separation of decarboxy-/dehydro- betalains byhigh-performance countercurrent chromatography in ion-pairsolvent systems

PPPP-18-18-18-18

Wang, G.; Chen, X.; Huang, X.;Yun, L.; Di, D.*

Development of new pattern PTFE column of HSCCC P-19P-19P-19P-19

Gomathi,C.; Boustie, J.; Audo,G.* ; Ismail, N. H.; Quémener, C.L.; Awang, K.

Multple dual mode centrifugal partition chromatography as anefficient method for the purification of 4-phenylcoumarinsform mesua elegans

PPPP-20-20-20-20

Weisz, A.*; Roque, J. A.; Mazzola,E. P.; Ito, Y.

Preparative separation of two subsidiary colors of FD & Cyellow No. 5 (Tartrazine) by spiral high-speed counter-currentchromatography

PPPP-21-21-21-21

Yang, Y.; Gu, D.; Chen, Q.; Yili,A.; Aisa, H. A.*

Novel sample pretreatment method for increasing thepreparative scale of pH-zone refining counter-currentchromatography

P-22P-22P-22P-22

Yang, Z.; Wu, S.*Solvent gradient counter-current chromatography purificationof podOPhyllotoxins from Dysosma versipellis (Hance)

PPPP-23-23-23-23

Yoo, G.; Kim, T. B.; Yang, H.;Park, J. H.; Kim, Y. C.; Sung, S. H.

Preparative separation of isoquinoline alkaloids from COPtisjaponica by high-speed counter-current chromatography

PPPP-24-24-24-24

Zhang, J.; Huang, X.; Pei, D.;Chen, X.; Di, D.*

Screening for anti-diabites fraction in the leaves of Oleaeuropaea L. by HPCCC

PPPP-25-25-25-25

Han,T.; Heuvel, R.; Sutherland, I.;Fisher, D.

Flow separation of cells in counter-current chromatography(CCC) centrifuges

PPPP-26-26-26-26

Erastov, A. A.; Yulya, A.;Zakhodjaeva, Y. A.; Voshkin, A. A.Kostanyan, A. E.*

Comparative study of three modifications of thecontrolled-cycle liquid-liquid chromatography device

PPPP-27-27-27-27


Authors Title Code

Qiu, F.; Friesen, J. B., McAlpine, J.B.; Lankin, D. C.; Pauli, G. F.*

Targeted analysis of natural products by K-basedcountercurrent separation

PPPP-28-28-28-28

van den Heuvel, R. N. A. M.;Harris, G.; Douillet, N.; Thickitt,C.; van den Heuvel, E. A.;Ignatova, S.

Characterisation of quaternary Heptane-Water basedphasesystems across the polarity rangeforCounter-currentChromatography

PPPP-29-29-29-29

Vieira, M. N.; Welke, K.;Murillo-Velásquez, J. A.;Leitão, S. G.; Winterhalter, P.*;Jerz, G.

Large-Scale Preparative Profiling of Anacardic Acids inCashew Nut Shell Oil Liquid by Spiral-Coil CountercurrentChromatography and Off-Line ESI-MS-MS ContinuousInfusion

PPPP-30-30-30-30

Bonsch, B.; Dillschneider, R.;Rosello, R.; Posten, C.;Winterhalter, P.*; Jerz, G....

Preparative Metabolite Profiling of the Diatom MicroalgaPhaeodactylum tricornutum by High-Speed CountercurrentChromatography and Continuous APCI-MS-MS Infusion

PPPP-31-31-31-31

Han, L. W.; Yuan, Y. Q.; He, Q. X.;Chen, X.Q.; Liu, K.C.*

Zebrafish bioassay-guided isolation of anti-angiogeniccomponents by high-speed counter-current chromatographyfrom licorice

P-3P-3P-3P-32222

Li,Y. l.*;Liu, Y. l.; Wang, P.; Chen,T.; Zhao, X.; Chen, C.; Sun ,J.

High-speed counter-current chromatography preparativeisolation and purification of four xanthone clycosides fromtibetan medicinal plant Halenia elliptica

P-3P-3P-3P-33333

Lin, X. J.; Dong, H. J.; Geng, Y. L.;Liu, F.; Wang, D. J.; Wang, X.*

A Combination of Ultrahigh pressure-Assisted Extraction withHigh-Speed Counter-Current Chromatography for thePreparation of Cantharidin from the blister beetle

P-3P-3P-3P-34444

Goll, J.; Frey, A.; Minceva. M.*Continuous separation of capsaicin and dihydrocapsaicin usingsequential centrifugal partition chromatography

PPPP-3-3-3-35555

Li, Y.*; Buonocore, F.; Barker, J.;Barton, S. J.; Sutherland, I.;Ignatova, S.

Separation of six ginsenosides from panax ginseng usinghigh-performance counter-current chromatography

PPPP-3-3-3-36666

Jiang, W.; Wu, B.* Isolation and Purification of Pheromones from Ground Beetlesby Counter-Current Chromatography

PPPP-3-3-3-37777


Authors Title Code

Ye, Q. X.; Yin, S.; Zhao, Z. M.;Yang, D. P.; Zhu, L. P.; Brown,L.; Wang, D. M.*

Separation of bioactive compounds from Dark tea usingHSCCC target-guided by α-glucosidase inhibitory activity

PPPP-3-3-3-38888

Gök, R.; Jerz, G.; Winterhalter, P.*Application of high speed countercurrent chromatography inorganic synthesis purification

PPPP----39393939

Okunji, C. O.; Awachie, P. I.;Iwu, M. M.; Tchimene, M.; Ito,Y.*

Preparative separation of biflavonoids from Garcinia Kolaheckel .seeds by spiral high-speed counter-currentchromatography

PPPP-4-4-4-40000

Xu, J.; Cao, X. L.*; Yin, L.; Cheng,C.; Ren, H.

Separation of anti-tumor constituents from KalOPanaxseptemlobus (Thunb.) Koidz by silica gel column andhigh-speed counter-current chromatography

PPPP-4-4-4-41111

Chen, Z. ; Lu, Y. B., *Effective and Preparative Separation of Bioactive Flavonoidsfrom Gynostemma pentaphyllum Tea Using Elution-ExtrusionCounter-Current Chromatography

PPPP-4-4-4-42222

Li, Z. H.; Li, M. H.*Preparative separation of polyphenols by high-speedcounter-current chromatography

PPPP-4-4-4-43333

Brownstein, K.; Rottinghaus, G.E.; Knight, M.; Ito, Y.; Folk, W. R.*

Isolation of the cycloartane glycosides from SutherlandiaFrutescens by HSCCC using the spiral tubing support column

PPPP-4-4-4-44444

Liu, D. H.; Shu, X. K.; Liu, W.;Wang, X.*; Yang, B.; Huang, L. Q.

Preparative separation of alkaloids from Aconitum carmichaeliby pH-zone-refining counter-current chromatography

PPPP-4-4-4-45555

Xu, M. X.; Liu, F.; Ma, X. L.;Zhao, H. Q.; Liu, J. H.; Wang, X.*

Preparative separation and purification of hypocrellins fromShiraia Bambusicola by high-speed counter-currentchromatography and preparative high performance liquidchromatoraphy

PPPP-4-4-4-46666

Gao, F. Y.; Fang, G.; Zhou, T. T.;Li, J.; Fan, G. R.*

Separation ,identification and purfication of active constituentsof Polygonum cuspidatum Sieb. et Zucc. byHPLC-PDA-ESI-MSn and HSCCC

PPPP-4-4-4-47777

Grudzien, L.; Fisher, D.; Madeira,L.; Ma, J.; Sutherland, I.; Garrard,I.

Purification of a small lectin from ahydroponic culture media usingcentrifugal partition chromatography

PPPP-4-4-4-48888

http://www.sciencedirect.com/science/article/pii/0021967394870764


app:ds: alkaloid


Authors Title Code

Duan, P. X.; Xu, Q. Q.; Zhang, L.;Wang, X. D.

Purification of luteim from kale by high speed countercurrentchromatography P-P-P-P-49494949

Michel, T.; Destandau, E.; Fougère,L.; Audo, G.; Elfakir, C.

New hyphenated CPC-HPLC-DAD-MS strategy forSimultaneous Isolation, Analysis and Identification ofPhytochemicals.

P-5P-5P-5P-50000

Chen, S. Q.; Zhou, T. T. Liu, C. C.;Fan, G. R.

Impurities preparaton and identification of sodium tanshinoneIIA sulfonate by HSCCC and LC-MSn P-5P-5P-5P-51111

Murhandini, S.; Keaveney, E.;Barton, S. J.;Barker, J.; Garrard,I.2;Fisher, D.; Ignatova, S.

Method optimisation for isolating purified α-mangostin fromgarcinia mangostana l. Rinds using HPCCC

P-5P-5P-5P-52222

Li, J. L.; Fang, L.; Wang, D. J.;Shu, X. K.; Geng, Y. L.; Wang, X.*

Preparative isolation and purification of biflavonoids fromephedra sinica by high-speed counter-current chromatography P-5P-5P-5P-53333

Wang, C.; Chao, Z.; Sun, W.;Wu, X.; Ito, Y. b

Isolation of Chemical Constituents from Ilex rotunda byHigh-Speed Counter-Current Chromatography P-5P-5P-5P-54444


P-1P-1P-1P-1PreparativePreparativePreparativePreparative separationseparationseparationseparation andandandand purificationpurificationpurificationpurification ofofofof chemicalchemicalchemicalchemical compositionscompositionscompositionscompositions fromfromfromfrom

MenthaMenthaMenthaMentha longifolialongifolialongifolialongifolia L.L.L.L. bybybyby normalnormalnormalnormal andandandand reversereversereversereverse phasephasephasephase high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-currentchromatographychromatographychromatographychromatography

YusupjanYusupjanYusupjanYusupjan EliEliEliEli a,ba,ba,ba,b,,,, YiYiYiYi YangYangYangYang aaaa,,,, AbulimitiAbulimitiAbulimitiAbulimiti YiliYiliYiliYili aaaa,,,, HajiHajiHajiHaji AkberAkberAkberAkber AisaAisaAisaAisa a,a,a,a,****a Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of

Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, Chinab 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:Keywords:Keywords: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 ofbiological samples with two-phase solvent systems [1-3]. However, the natural products or their fractions areconstituted by complex mixtures. Usually, the studies focus on separation of compounds in the crude fraction withbioactivity from herb plant. In this fraction, someconstituents are hydrophobic and some are hydrophilic.When the normal phase HSCCC was used for separationof hydrophobic compounds with appropriate K values, theretention time of hydrophilic compounds were very bigbecause of the large K values, and vice versa. So normaland reverse phase HSCCC can be combined for thecomprehensive investigation of chemical compositions inthe active fraction. Mentha longifolia L. is a traditionalUighur medicine for the treatment of gastritis, carminative,headache, curette, sinusitis, hemorrhoids, diuretic,menstruate etc [4]. However, scientific data concerning thebasic composition are lacking. It encouraged us to performthe phytochemical investigation for Mentha longifolia L.In the study, five compounds were isolated from Menthalongifolia L. by normal and reverse phase HSCCC. Thenormal and reverse phase HSCCC separations wereperformed with two biphasic solvent systems composed ofchloroform-methanol-water (4:3:2, v/v) andn-hexane-ethyl acetate-methanol-water-acetic acid(1:10:1:10:0.25, v/v), respectively. As a result, 11.8mg ofdiosmetin, 5.3mg of acacetin and 4.6mg of apigenin wereobtained from 200 mg ethyl acetate extracts of Menthalongifolia L. in normal phase elution mode by HSCCC.And 6.1 mg of rosmarinic acid and 3.2 mg caffeic acidwere obtained from 200 mg ethyl acetate extracts ofMentha longifolia L in reverse phase elution mode byHSCCC. Their structures （Fig. 1）were identified by MSand NMR.ReferencesReferencesReferencesReferences

To be ordered numerically following the appearance in the text with this format: Arial normal letter font 8. No spacebetween references. Citation style follows JACS/ACS format, an Endnote style file can be obtained fromwww.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.

Figure 1. The structures of isolated compoundsfrom Mentha longifolia L. by normal and reversephase high-speed counter-currentchromatography.


P-2P-2P-2P-2PPPPreparativereparativereparativereparative separationseparationseparationseparation andandandand purificationpurificationpurificationpurification ofofofof fivefivefivefive oxindoleoxindoleoxindoleoxindole alkaloidsalkaloidsalkaloidsalkaloids fromfromfromfromgelsemiumgelsemiumgelsemiumgelsemium eleganseleganseleganselegans bybybyby ph-zone-refiningph-zone-refiningph-zone-refiningph-zone-refining counter-currentchromatographycounter-currentchromatographycounter-currentchromatographycounter-currentchromatography

LeiLeiLeiLei Fang,Fang,Fang,Fang, JiaLianJiaLianJiaLianJiaLian Li,Li,Li,Li, YunLiangYunLiangYunLiangYunLiang Lin,Lin,Lin,Lin, JieJieJieJie Zhou,Zhou,Zhou,Zhou, YangLingYangLingYangLingYangLing Geng,Geng,Geng,Geng, XiaoXiaoXiaoXiao Wang*Wang*Wang*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:Keywords:Keywords: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 beenused traditionally for the treatment of pain, spasticity, and skin ulcers in Chinese folkloric medicine (1). Thepharmacologically active constituents of G. elegans mainly consist of oxindole alkaloids, and more than 70alkaloids based on six different structural skeletons have

been isolated from G. elegans (2,3).These alkaloids demonstrate potent cytotoxic, immunomodulating andantiarrhythmic, analgesic, and anti-inflammatory activities (4,5). Considering such various biologicallyactivities of oxindole alkaoloids, it is necessary to develop an efficient method to separate and purify largequantities of single oxindole alkaoloid with high purity for further pharmacological research.

In this research, pH-zone-refining counter-current chromatography was successfully applied to thepreparative separation and purification of five oxindole alkaloids from the stems of G. elegans. The

experiment was performed with a two-phase solventsystem composed of petroleum

ether–ethyl acetate–methanol–water (3:7:1:9, v/v),where 10mM triethylamine (TEA) was added to the

upper organic stationary phase as a retainer and10mM hydrochloric acid (HCl) to the aqueousmobile phase as an eluter. 3.0 g of the total alkaloidwas purified in one-step separation, yielding 110 mgof 19-xo-gelsenicine, 126 mg of gelsemine, 302 mgof gelsevirine, 107 mg of gelsenicine and 89 mg ofhumantenine with the purities of 98.1%, 98.5%,98.7%, 98.4% and 98.8%, respectively, asdetermined by high-performance liquidchromatography. The structures of the oxindolealkaloids were identified by UV, ESI-MS, 1H NMRand 13C NMR.

This is the first report that oxindole alkaloids from the stems of G. elegans were successfully isolated andpurified by the pH-zone-refining counter-current chromatography method, which demonstrated thatpH-zone-refining CCC is a fast, effective and powerful technique for the isolation and purification of alkaloidsfrom natural herbs.


1. Editorial committee of Chinese Materia Medica. Chinese Materia Medica, Vol.17; Shanghai Science and Technology Press: Shanghai, 1999199919991999; p.2132. Xu, Y. K.; Yang, S. P.; Liao, S. G.; Zhang, H.; Ling, L. P.; Ding, J. J.; Yue, M. J. Nat. Prod. 2006200620062006, 69, 1347–1350.3. Kogure, N.; Ishii, N.; Kitajima, M.; Wongseripipatana, S.; Takayama, H. Org. Lett. 2006200620062006, 8, 3085–3088.4. Rujjanawate, C.; Kanjanapothi, D., Panthong, A. J. Ethnopharmacol. 2003200320032003, 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. 2006200620062006, 69, 715–718.

Figure 1. Separation of six oxindole alkaloids fromthe stems of G. elegans by pH-zone-refining CCC.Solvent system: petroleum ether–ethylacetate–methanol–water; sample size: 3.0 g;flow-rate: 1.5 ml/min; detection: 254 nm; revolutionspeed: 800 rpm. a: 19-xo-gelsenicine; b: gelsemine;c: gelsevirine; d: gelsenicine; e: humantenine



P-3P-3P-3P-3EvaluationEvaluationEvaluationEvaluation ofofofof LimoneneLimoneneLimoneneLimonene asasasas aaaa possiblepossiblepossiblepossible "green""green""green""green" nonnonnonnon polarpolarpolarpolar solventsolventsolventsolvent forforforfor alkanealkanealkanealkane

replacementreplacementreplacementreplacementKarineKarineKarineKarine Faure,Faure,Faure,Faure, NazimNazimNazimNazim Mekaoui,Mekaoui,Mekaoui,Mekaoui, AlainAlainAlainAlain BerthodBerthodBerthodBerthod

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 bypetroleum industry, alkanes are non renewable and pollutant. To develop ecofriendly technologies, the policy isto favor bio-renewable solvents. Limonene is, with the possible exception of α-pinene, the most frequentlyoccurring natural monoterpene. It is a major constituent (90-95%) of the orange essential oil. Since it comesfrom food waste, its use in industry is highly plebiscited.

Following Friesen [1] that first proposed Limonene as a possible renewable solvent in CCC liquid systemformulations, limonene was tested for the replacementof heptane in the simple solvent systemheptane/methanol/water.

The Friesen initial results were confirmed: in hydrodynamic instruments, the very low density differencebetween limonene (lower phase) and the methanol/ water mixture resulted in a constant flooding and poor ornearly no phase retention (small Sf). The use of limonene and heptane blends could help but it conflicts withthe environmental concern.

The new finding is that hydrostatic instruments (centrifugal partition chromatographs) could easily retainedlimonene as stationary phase, with Sf reaching values as high as 60%. The low density difference is actuallyan advantage leading to reduced working pressures (around 15 bars), allowing for the user to work at high rotorrotation 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. Theoverall 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 tomodulate 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.

ReferenceReferenceReferenceReference

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,

Figure 1. Limonene represents90-95% of orange essential oil

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


P-4P-4P-4P-4NovelNovelNovelNovel designdesigndesigndesign forforforfor centrifugalcentrifugalcentrifugalcentrifugal counter-currentcounter-currentcounter-currentcounter-current chromatography:chromatography:chromatography:chromatography: Ⅵ.... EllipsoidEllipsoidEllipsoidEllipsoid

columncolumncolumncolumnDongyuDongyuDongyuDongyu GuGuGuGu a,a,a,a, bbbb,,,, YiYiYiYi YangYangYangYang a,a,a,a, bbbb,,,, HajiHajiHajiHaji AkberAkberAkberAkber AisaAisaAisaAisa bbbb,,,, YoichiroYoichiroYoichiroYoichiro ItoItoItoIto a,a,a,a,****

a Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and BloodInstitute, National Institutes of Health, Bethesda, MD 20892, USA

b Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute ofPhysics 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, USAKeywords:Keywords:Keywords:Keywords: ellipsoid column, hydrostatic counter-current chromatographic system, centrifugal counter-currentchromatography, retention of the stationary phase, peak resolution

In our previous studies, a series of novel column designs has been introduced to further improve theperformance of Hydrostatic counter-current chromatography (CCC) which can produce highly efficientanalytical 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 mountedparallel to the radially acting centrifugal force field. The performance of the system was examined in varioustest samples [6,7]. The high partition efficiency of this system may be explained by hydrodynamic motion andinteraction of the two phases in the column at various angles. When the column angle is set at 0º against theradially acting centrifugal force field, the second loop of figure-8 provides two separate partition segments fordroplet flow to improve Rs (Fig. 1A) [7]. Based on this finding, the ellipsoid column is designed in the presentstudy, which can provide a much longer partition segment than that in the figure-8 column (Fig. 1B). Theperformance of the ellipsoid column with a capacity of 3.4 mL was examined with three different solventsystems composed of 1-butanol-acetic acid-water (4:1:5, v/v) (BAW), hexane-ethyl acetate-methanol-0.1 MHCl (1:1:1:1, v/v) (HEMH), and 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate inwater (PEG-DPP) each with suitable test samples. In dipeptide separation with BAW system, both stationaryphase retention (Sf) and Rs of the ellipsoid column were much higher at 0º column angle than at 90º columnangle, where elution with the lower phase at a low flow rate produced the best separation yielding Rs at 2.02with 27.8% Sf at a flow rate of 0.07 ml/min. In the DNP-amino acid separation with HEMW system, the bestresults were obtained at a flow rate of 0.05 ml/min with 31.6% Sf yielding high Rs values at 2.16 betweenDNP-DL-glu and DNP-β-ala peaks and 1.81 between DNP-β-ala and DNP-L-ala peaks. In protein separationwith PEG-DDP system, lysozyme and myolobin were resolved at Rs of 1.08 at a flow rate of 0.03 ml/min with38.9% Sf. Most of those Rs values exceed those obtained from the figure-8 column under similar experimentalconditions previously reported.


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. A1218(2011)6128.7. Y. Yang, D. Gu, H. A. Aisa, Y. Ito, J Chromatogr. B879(2011)3802.

Figure 1. Hydrodynamic motion and interaction of the twophases in the figure-8 and ellipsoid column. The column wasparallel to the acting centrifugal force


P-5P-5P-5P-5AAAA newnewnewnew two-phasetwo-phasetwo-phasetwo-phase solventsolventsolventsolvent systemsystemsystemsystem ofofofof chloroform/acetylchloroform/acetylchloroform/acetylchloroform/acetyl acetate/methanol/wateracetate/methanol/wateracetate/methanol/wateracetate/methanol/water

andandandand itsitsitsits useuseuseuse inininin HSCCCHSCCCHSCCCHSCCC separationseparationseparationseparation ofofofof aconitinesaconitinesaconitinesaconitines fromfromfromfrom aconiteaconiteaconiteaconite rootsrootsrootsrootsQuan-BinQuan-BinQuan-BinQuan-Bin Han*Han*Han*Han*

School of Chinese Medicine, Hong Kong Baptist University*[email protected]

Keywords:Keywords:Keywords:Keywords: two-phase solvent system， HSCCC， aconitines

High-speed counter-current chromatography (HSCCC) has been popularly used to prepare high-purity naturalcompounds in laboratories [1,2]. The unique mechanism makes it a magic and powerful separation technique inwhich two-phase solvent system plays a crucial role. Basically, HSCCC is a continued liquid-to-liquid solventpartition where the target compounds are competitively distributed between the two-phase solvents due to theirdifferent partition coefficients (K values). The competition reaches a climax when K value is 1 which means thetwo-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 stepwiseelution; 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 of2.75:1:1.5:2 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 naturalcompounds [3]. Aqueous two-phase solvent systems like were des igned for macromolecules [4-9]. Regarding theseparation of small molecules, two-phase solvent systems composed of organic solvents and water are commonlyused, for example, the most popular system hexane/acetyl acetate/methanol/water [3]. The lower phase is alwaysaqueous. Of course, there is an exception, namely chloroform/methanol/water in which the lower phase is organicsolvent because chloroform contains higher density.

Selection of a proper two-phase solvent system is definitely the most important step in a successful HSCCCseparation. At the same time, it is also time consuming. It is hard to make water and oil contain similar dissolvingcapacities. We reports in this paper a new two-phase solvent system in which both phases can contain sufficientorganic solvents to balance their dissolving capacities. Its two-phase forms based on not only the water-oil differencebut also the difference of density. It is chloroform/acetyl acetate/methanol/water. Different fromchloroform/methanol/water, the upper phase contains more organic solvent acetyl acetate. Its polarity is close tochloroform, 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 usedhexane/acetyl acetate/methanol/water failed.


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 and4. 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) 5930.10.10.10.10. Z.G. Jiang, Q.Z. Du, L.Y. Sheng, Fenxi Huaxue 37 (2009), 412.


P-6P-6P-6P-6IsolationIsolationIsolationIsolation andandandand purificationpurificationpurificationpurification ofofofof ecdysteroneecdysteroneecdysteroneecdysterone fromfromfromfrom SerratulaSerratulaSerratulaSerratula chinensischinensischinensischinensis S.S.S.S. MooreMooreMooreMoore bybybyby

high-speedhigh-speedhigh-speedhigh-speed countercountercountercounter currentcurrentcurrentcurrent chromatographychromatographychromatographychromatographyJieyunJieyunJieyunJieyun HuangHuangHuangHuang1111,,,, XinjunXinjunXinjunXinjun Xu*,Xu*,Xu*,Xu*, ZhishengZhishengZhishengZhisheng XieXieXieXie1111,,,, MeiMeiMeiMei YangYangYangYang1111,,,, ChunyanChunyanChunyanChunyan XieXieXieXie1111

SchoolSchoolSchoolSchool ofofofof PharmaceuticalPharmaceuticalPharmaceuticalPharmaceutical Sciences,Sciences,Sciences,Sciences, SunSunSunSun Yat-Yat-Yat-Yat-SSSSenenenen UniversityUniversityUniversityUniversity* No. 132, East Waihuan Rd.,Guangzhou Higher Education Mega Center, 510006 Guangzhou, China

e-mail address: [email protected]. fax: +86 020 39943041.

KeKeKeKeywords:ywords:ywords:ywords: Serratula chinensis S. Moore, HSCCC, ecdysterone

Serratula chinensis S. Moore (Guang Sheng Ma) is a perennial herbaceous plant growing mainly in SouthChina [1]. Ecdysterone is the major component of Serratula chinensis S. Moore [2]. In this paper, ecdysterone wasisolated and purified from Serratula chinensis S. Moore by high-speed counter-current chromatography with asolvent system of ethyl acetate-n-butanol-water (4:1:5, v/v/v) in one step. From 200 mg of the n-butanol extract ofSerratula chinensis S. Moore, 23 mg of ecdysterone was obtained with purity of 99.3%, as determined by HPLC. Thestructure was identified by MS, UV, and NMR analysis. In this study, a rapid method for isolation and purification ofecdysterone 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 thethe polarity of ecdysterone, n-butanol-water was used to test the K value firstly. But the K value measured by HPLCof n-butanol-water was too high, indicating that it was not a suitable system for the separation. So ethyl acetate wasadded 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 differentvolume ratios were tested. No.2 and No.3 was not suitable because the K values were still high. Experiments showedthat No.4 could efficiently separate ecdysterone. The K value of No.4 was 0.90 and it was very suitable to separatethe target compound 23 mg of ecdysterone was obtained from 200 mg of Serratula chinensis S. Moore n-butanolextract. Therefore, No.4 was chose as the solvent system for HSCCC.

No. Solvent system Ratio(v/v/v)

K

No.1 n-butanol-water 1:1 5.03

No.2Ethyl

acetate-n-butanol-water

2:3:5 3.93

No.3Ethyl


3:2:5 2.48

No.4Ethyl


4:1:5 0.90


1. Ling, T. J.; Xia, T.; Wan, X. C.; Li, D. X.; Wei, X. Y.. Cerebrosides from the roots of Serratula chinensis. MOLECULES. 2006200620062006, 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. 2011201120112011, 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 deserticolaby high-speed counter-current chromatography. Food Chemistry. 2008200820082008, 108, (2), 702-710.

Table 1. The K value of the targeted component

measured in different solvent systems

Figure1. HSCCC chromatogram of the n-butanol extract ofSerratula chinensis S. Moore. The column volume was 120 mL.Solvent system: ethyl acetate-n-butanol -water (4:1:5, v/v/v). Theupper phase served as the stationary phase. The lower phase (mobilephase) was pumped into the column from the head-to-tail at a flowrate of 2 mL·min-1. The rotation speed was 860 rpm. Retention ofthe stationary phase: 54.2%. Injection volume: 10 mL; thewavelength: 254 nm; fraction collector: 3 min for each tube.



P-7P-7P-7P-7DDDDual-modeual-modeual-modeual-mode counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography appliedappliedappliedapplied totototo isolationisolationisolationisolation maslinicmaslinicmaslinicmaslinic andandandand

oleanolicoleanolicoleanolicoleanolic acidsacidsacidsacids fromfromfromfrom thethethethe oliveoliveoliveolive millingmillingmillingmilling subproductsubproductsubproductsubproductHuangHuangHuangHuang Xin-YiXin-YiXin-YiXin-Yi1111,,,, ChenChenChenChen Xiao-FenXiao-FenXiao-FenXiao-Fen1,21,21,21,2,,,, PeiPeiPeiPei DongDongDongDong1111,,,, ZhangZhangZhangZhang JiaJiaJiaJia1111,,,, DiDiDiDi Duo-LongDuo-LongDuo-LongDuo-Long1111****

1Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for NaturalMedicine 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]: Dual-mode counter-current chromatography, maslinic acid, oleanolin acid

Maslinic acid (MA) and oleanolic acid (OA) are pentacyclic triterpene derivatives which have recently caused greatinterest due to their many biological effects, such as antioxidant activity, anti-HIV activity, anti-inflammatory andsupressive effects on glycogen phosphorylase, cancer cell, hyperlipidemia, and so on. Olive fruit has high contents ofMA 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 classicalsolvent system consist of n-hexan-ethyl acetate–methanol –water (HEMW) was used to achieve this separation. Aseries of HEMW biphasic systems with different composition were screened to optimize the solvent system for CCCseparation and their partition coefficients (k) values of the target compounds were measured and summarized inTable 1.

Given the enormous range of k values of MA and OA, the dual-mode (DM) was used to elute the twocompoents. The separation was achieved on a DE Spectrum HPCCC under analysis condition. The upper phase ofbiphasic system was as stationary phase and moble phase was bumped at a flow rate of 1.0 mL/min with rotation at1500 rpm and at RP mode. When the MA was eluted, the elution modes were changed from initial RP to NP byswitching the positionof the valves. The effluent was collected each minute and analysed by HPLC. The both puritiesof MA and OA were more than 90%.

Table 1. The k values of MA and OA in different solvent systems.

SolventSolventSolventSolvent systemsystemsystemsystem

compositioncompositioncompositioncomposition (v:v)(v:v)(v:v)(v:v)

kkkk valuevaluevaluevalue

MAMAMAMA OAOAOAOA

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



P-8P-8P-8P-8PPPPreparativereparativereparativereparative separationseparationseparationseparation ofofofof majormajormajormajor CCCConstituentsonstituentsonstituentsonstituents inininin OOOOpuntiapuntiapuntiapuntia ficus-indicaficus-indicaficus-indicaficus-indica bybybyby

high-speedhigh-speedhigh-speedhigh-speed countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatographyTae 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] 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-speedcountercurrent chromatography. The two-phase solvent systems comprising methylenechloride-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 toHSCCC. Isolated compounds were elucidated by ESI-MS and NMR techniques. The purity of compounds wasevaluated to be over 90 % by HPLC-DAD.

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

SolventSolventSolventSolvent systemsystemsystemsystem KKKK valuevaluevaluevalue

CHCl3-MeOH-H2O(4:1:2, v/v/v) 19.16CHCl3-MeOH-H2O (3:4:2, v/v/v) 4.25CH2Cl2-MeOH-BuOH-H2O (4:4:3, v/v/v) N/ACH2Cl2-MeOH-BuOH-H2O (3:1:1:3, v/v/v/v) 2.26CH2Cl2-MeOH-BuOH-H2O (3:1:2:3, v/v/v/v) 2.10CH2Cl2-MeOH-BuOH-H2O (3:2:1:3, v/v/v/v) 2.90CH2Cl2-MeOH-BuOH-H2O (5:4:3:5, v/v/v/v) 1.33Figure1. Separation diagram

elucidated by ESI-MS and NMR techniques.The purity of compounds was evaluated to beover 90 % by HPLC-DAD.

Figure 2. Major flavonoids in Opuntia ficus-indica

Figure 3. HSCCC chromatogram of Opuntiaficus-indica

Figure 4. HPLC-DAD chromatogram offlavonoids in Opuntia ficus-indica


1. Lee, E. H.; Kim, H. J.; Song, Y. S.; Jin, C.; Lee,K.; Cho, J.; Lee, Y. S. Arch. Pharm. Res. 2003200320032003, 26.

2. Jeong, S.J.; Jun, K. Y.; Kang, T. H.; Ko, E. B.;Kim, Y. C. Saengyak Hakhoechi. 1999199919991999, 30.



P-9P-9P-9P-9

EliminationEliminationEliminationElimination ofofofof metalmetalmetalmetal elementselementselementselements bybybyby high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographybybybyby introducingintroducingintroducingintroducing airairairair andandandand solidsolidsolidsolid absorbentsabsorbentsabsorbentsabsorbents

EiichiEiichiEiichiEiichi KitazumeKitazumeKitazumeKitazume1,*1,*1,*1,*,,,, TomoakiTomoakiTomoakiTomoaki SudoSudoSudoSudo1111,,,, HanaeHanaeHanaeHanae KuboKuboKuboKubo1111,,,, MikiMikiMikiMiki WatanabeWatanabeWatanabeWatanabe1111 YoichiroYoichiroYoichiroYoichiro ItoItoItoIto2222

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:Keywords:Keywords:Keywords: counter-current chromatograph, enrichment, metal elements

Radioactive materials were widely spread in the atmosphere from the nuclear power plant inFukushima, Japan, following the strong earthquake on March 11, 2011. Now, cesium 137 with ahalf-life of 30 years especially poses a problem. Since a large area of soil and water has beenpolluted near the nuclear power plant, immediate measures for decontamination are urgentlyneeded. Although there are various decontamination methods, for example by washing buildingsand their roofs with high-pressure water-drainage equipment, the contamination is only washedaway 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 nowhow to store or dispose the removed top-soil. Ideally the best method would be to condense thereleased radioactive materials into one spot and (if possible) get Ali Baba’s Genie to return thingsto their original state. Yet, this is far from realistic.

Although high-speed counter-current chromatography (HSCCC) has so far been used tocontinuously condense impurities in a liquid phase without dispersing them, the method may alsobe used for efficient decontamination of metal elements including radioactive materials. Then, itmay be possible to operate HSCCC with air, liquids and particles in the column in order tocondense 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 orair and adsorbent (CDP silica or cyclodextrin wrapped in a silica skeleton) in a column. Theeffectiveness of this novel HSCCC method is investigated by continuously introducing watercontaining metal elements (which simulates contaminated water) through the column.


P-10P-10P-10P-10PreparativePreparativePreparativePreparative isolationisolationisolationisolation ofofofof chemicalchemicalchemicalchemical constituentsconstituentsconstituentsconstituents fromfromfromfrom thethethethe rootsrootsrootsroots ofofofof PhlomisPhlomisPhlomisPhlomis

umbrosaumbrosaumbrosaumbrosa bybybyby high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography

KyoungKyoungKyoungKyoung JinJinJinJin Lee,Lee,Lee,Lee, InInInIn JinJinJinJin HaHaHaHa andandandand YeongYeongYeongYeong ShikShikShikShik KimKimKimKim

Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul151-742, Korea

**Fax: +82 2 765 4768599. E-mail: [email protected] (Prof. Yeong Shik Kim)

Keywords:Keywords:Keywords: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.



P-11P-11P-11P-11Bioassay-guidedBioassay-guidedBioassay-guidedBioassay-guided separationseparationseparationseparation andandandand purificationpurificationpurificationpurification ofofofof water-solublewater-solublewater-solublewater-soluble antioxidantsantioxidantsantioxidantsantioxidants

fromfromfromfrom CarthamusCarthamusCarthamusCarthamus TinctoriusTinctoriusTinctoriusTinctorius L.L.L.L. bybybyby combinationcombinationcombinationcombination ofofofof chromatographicchromatographicchromatographicchromatographic techniquestechniquestechniquestechniquesLingnanLingnanLingnanLingnan LiLiLiLi a,a,a,a, bbbb,,,, YiYiYiYi YangYangYangYang aaaa,,,, XuelingXuelingXuelingXueling HouHouHouHou aaaa,,,, HangHangHangHang BaBaBaBa aaaa,,,, DongyuDongyuDongyuDongyu GuGuGuGu aaaa,,,,

RahimaRahimaRahimaRahima AbdullaAbdullaAbdullaAbdulla aaaa,,,, XueleiXueleiXueleiXuelei XinXinXinXin aaaa,,,, GuirongGuirongGuirongGuirong WuWuWuWu bbbb,,,, HajiHajiHajiHaji AkberAkberAkberAkber AisaAisaAisaAisa a,a,a,a, ****a Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of

Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, Chinab 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:Keywords:Keywords: Bioassay-guided separation, combination of chromatographic techniques, high-speedcounter-current chromatography, Carthamus tinctorius L., water-soluble antioxidants.The flowers of Carthamus tinctorius L. (safflower), have been used as Chinese traditional medicine for over2500 years and for the treatment of stroke, coronary heart disease, and angina pectoris [1,2]. In our study, theextract of safflower showed the antioxidant activity prompted us to investigate the chemical constituents and itsassociation with antioxidant. But most of active compounds with high hydrophilicity in the active fraction areminor ingredients. The simultaneous preparation of major compounds and separation of these minor activecompositions is a very big challenge, especially for water-soluble minor compositions. In order to investigatethe minor water-soluble antioxidants, in the present study, a combined chromatographic method usinghigh-speed counter-current chromatography (HSCCC) which has been widely used for various naturalcompounds separation [3,4] and Sephadex LH-20 chromatography was established for bioassay-guidedseparation and purification of water-soluble antioxidants from of Carthamus tinctorius L. florets. Following aninitial cleanup step on the AB-8 macroporous resin, the crude sample Ⅱ was obtained and exhibited a potentialABTS radical cation scavenging activity with the SC50 value of 49.28 μg mL-1, which was separated byHSCCC. The HSCCC separation was performed with a two-phase solvent system composed ofn-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 hydroxysaffloryellow A (HSYA) with 98% purity, a major component, was separated from 20 mg crude sample Ⅱ. Toincrease the yield of HSYA and investigate the minor compositions, the sample size of HSCCC separation wasenlarged. Crude sample Ⅴ composed of HSYA was obtained. To further increase purity of HSYA and removethe acid which maybe destroyed the natural antioxidants, the Sephadex LH-20 chromatography was employedand eluted by distilled water at the flow rate of 1.5 mL min-1. The separation yielded 184 mg HSYA (1), 10 mg5,4’-dihydroxyflavone-3,6-di-O-β-D-glucoside-7-O-β-D-glucuronide (2), 3.0 mg chlorogenic acid (3), and 3.2mg 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 byABTS 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


1. Li D; Mündel H. Safflower, Carthamus tinctorius L. Bioversity International, Rome, 1996199619961996, p. 8-9.2. Suleimanov T A, Chem. Nat. Compd. 2004200420042004, 40, 13-15.3. Berthod A; Maryutina T; Spivakov B; Shpigun O; Sutherland I A. Pure Appl. Chem. 2009200920092009, 81, 355-387.4. Ito, Y. J. Chromatogr. A 2005200520052005, 1065, 145-168.


P-12P-12P-12P-12PPPPreparativereparativereparativereparative isolationisolationisolationisolation andandandand purificationpurificationpurificationpurification ofofofof tanshinonestanshinonestanshinonestanshinones fromfromfromfrom salviasalviasalviasalvia miltiorrhizamiltiorrhizamiltiorrhizamiltiorrhizabungebungebungebunge bybybyby aaaa novelnovelnovelnovel counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography withwithwithwith anananan uprightuprightuprightupright conicalconicalconicalconical

coilscoilscoilscoils

JunlingJunlingJunlingJunling LiangLiangLiangLiang1111,,,, JieJieJieJie MengMengMengMeng1111,,,, ShihuaShihuaShihuaShihua WuWuWuWu*,1*,1*,1*,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].

KeyKeyKeyKey words:words:words:words: counter-current chromatography, conic holder hub, Salvia miltiorrhiza Bunge, Tanshinones

AbstractAbstractAbstractAbstract Counter-current chromatography (CCC) has been playing a critical role in the separation of naturalproducts, which has been recognized as an important source of pharmaceutical agents. Since CCC was firstinvented 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 includinghigher retention of stationary phase, and solutes resolution than other CCC coils， such as helical and toridalcoils. However, these coils both are distributed in one- or two-dimensional centrifugal force field except thecross-axis CCC which holds a three-dimensional centrifugal field. This work introduced a novel conical CCCwith upright conic coils, which was wound on three identical upright conical holder hubs by head-to-tail and left-handdirection (Fig.1Fig.1Fig.1Fig.1). Compared with helical and spiral CCC, conical CCC holds a special centrifugal force gradientboth in axial and radial direction enabling the CCC much higher resolution. And in details, it enables different linearvelocity not only in the same layer of column but also between different layers, and large volume of columnand sample loop which make higher sample loading capacity. In this work, the novel CCC was successfullyapplied to isolate and purify danshinones from crude extract of Salvia miltiorrhiza Bunge. As shown in Fig.Fig.Fig.Fig. 2222,higher resolution has been obtained compared with traditional CCC apparatus by the solvent system ofhexane-ethyl acetate –ethanol-water with the volume ratio of 5:5:7:3. As a result, 4 tanshinones were wellresolved from 500 mg to 1g crude samples.

Fig.1Fig.1Fig.1Fig.1 The design principle of upright conicholder hub system on the novel CCCapparatus.

Fig.2Fig.2Fig.2Fig.2 Chromatographs of traditional CCCapparatus (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) and500mg (B) with the rotation speed of 900rpm (A) and 600rpm (B).

1+2+31+2+31+2+31+2+3 (A)(A)(A)(A)

44441111 2222 3333

(B)(B)(B)(B)



P-13P-13P-13P-13PPPPreparativereparativereparativereparative separationseparationseparationseparation ofofofof threethreethreethree flavoniodsflavoniodsflavoniodsflavoniods fromfromfromfrom thethethethe flowerflowerflowerflower budsbudsbudsbuds ofofofof daphnedaphnedaphnedaphne

genkwagenkwagenkwagenkwa bybybyby ph-zone-refiningph-zone-refiningph-zone-refiningph-zone-refining vortexvortexvortexvortex counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography

YongYongYongYong LiangLiangLiangLiang1,21,21,21,2 YingYingYingYing MaMaMaMa1111 YoichiroYoichiroYoichiroYoichiro ItoItoItoIto1*1*1*1*

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,ChinaYong Liang address: Phone number: 8620-13539873265, E-mail: [email protected]

*Correspondence address: Fax: 301-402-0013; E-mail:[email protected]:Keywords:Keywords:Keywords: flavonoids, Daphne genkwa, pH-zone-refining vortex counter-current chromatography, High-speedcounter-current chromatography, preparation separation

Daphne genkwa Sieb. et Zucc. (Yuanhua in Chinese), a well- known traditional Chinese medicinal herb listed in the ChinesePharmacopoeia [1], has anti-inflammation, anti-leukemia, and anti-tumor activities [2]. Chemical investigations have disclosedthat 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 forfurther pharmacological studies and new drug exploration.pH-zone-refining CCC [4] is generally employed as a large-scale preparative technique for separating ionizableanalytes. 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 inthe 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, itwas selected as the two-phase solvent system for the pH-zone- refining vortex CCC separation. The method used inpH-zone-refining vortex CCC was based on D. genkwa characteristic acidity which is determined by the number of phenolichydroxyl groups and their positions in the molecule. Among those target compounds, apigemin is the most acidic, followed by3-hydroxy-genkwanin and genkwanin in this order. The apigemin could be eluted with the ammonium hydroxide mobile phaseused in the ordinary pH-zone-refining system. However, other two target compounds could not be eluted with ammoniumhydroxide in the mobile phase since these weakly acidic phenol groups do not form stable salts with ammonium hydroxide. Thisproblem was overcome using a stronger base, such as NaOH or Na2CO3 as an eluter in the aqueous mobile phase. Therefore, aseries of different kinds of bases such as NH3, NaOH and Na2CO3 and their concentrations were tested as an eluter in the aqueousmobile phase. The experiment proved that 10 mM Na2CO3 was the most suitable eluter in the mobile phase combined with 10mM TFA (trifluoroacetic acid) as a retainer in the stationary phase for separation of apigemin, 3-hydroxy-genkwanin andgenkwanin. The reason may be that Na2CO3 is a kind of diacidic base, and it produces two pH changes in the process of itsdissociation.

Reference1. China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, Beijing, China 2010:1092. Lee, M. Y., Park, B. Y., Kwon,,etc, Int. Immunopharmacol.9(2009) 878–8853. V.P. Androutsopoulos, K. Ruparelia, R.R.J. Arroo, A.M. Tsatsakis, D.A. Spandidos, Toxicology 264 (2009) 1624. Yoichiro ito, J. chromatogr. A 1065 (2005) 145–16


P-14P-14P-14P-14MultipleMultipleMultipleMultiple DualDualDualDual ModeModeModeMode andandandand trappingtrappingtrappingtrapping moleculesmoleculesmoleculesmolecules forforforfor CoomassieCoomassieCoomassieCoomassie BrilliantBrilliantBrilliantBrilliant BlueBlueBlueBlue

G-250G-250G-250G-250 purificationpurificationpurificationpurificationNazimNazimNazimNazim MEKAOUI*,MEKAOUI*,MEKAOUI*,MEKAOUI*, KarineKarineKarineKarine FAUREFAUREFAUREFAURE andandandand AlainAlainAlainAlain BERTHODBERTHODBERTHODBERTHOD

Institut des Sciences Analytiques, Université de Lyon, CNRS, Villeurbanne, France* E-mail : [email protected] ; Fax : +33 472 448 319

Keywords:Keywords:Keywords: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 canbe achieved without peak distortion (non linearity due to overload). The reason is that compounds with simplesolution equilibrium have access to a large stationary phase volume in liquid-liquid contactors compared toadsorption 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 realcountercurrent motion of two non-miscible phases [2]. However, separations with this approach use specificallydesigned 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 ascendingand 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 binaryseparations [4]. In the current work, this separation method is applied to the purification of a commercial dyeof Coomassie Brilliant Blue G-250 (CBB), which is required a higher purity when used as an indicator ofamine content in biological material.

The liquid system heptane/1-butanol/water 2:3:4 (v/v) was found satisfactory to separate crude CBB in threegroups of components. Firstly a polar group, partitioning in the aqueous lower phase, then an intermediateone, partitioning well between the aqueous and organic phases, and finally an apolar group preferring theorganic phase. The dual-mode way was convenient by injecting the crude CBB in the middle of a two coilanalytical CCC instrument. The multi dual-mode purification was performed allowing to get rid of the polarimpurities in the aqueous phase at the column tail and of the apolar ones in the organic phase at the columnhead. The intermediate polarity fraction contained the desired dyes without impurities and trapped inside theCCC 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 CBBwere trapped inside the CCC column obtained from 1 g of crude CBB in 3 h using less than 250 mL of organicsolvents. The purified CBB was finally collected at the end of the experiment by simple extrusion. It gavetotal satisfaction in testing polyclonal antibodies adsorbed onto a monolithic support.


1. A. Berthod, Countercurrent chromatography: the support-free liquid stationary phase, Analytical Chemistry Series, Volume38, Elsevier, Amsterdam, 2002.2002.2002.2002.2. R. van den Heuvel, B. Mathews, S. Dubant, I.A. Sutherland, J. Chromatogr. A, 2009, 1216121612161216, 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, 1127112711271127, 45.4. N. Mekaoui, A. Berthod, J. Chromatogr. A, 2011, 1218,1218,1218,1218, 6061-6071.


P-15P-15P-15P-15Multi-dimensionMulti-dimensionMulti-dimensionMulti-dimension counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography forforforfor high-high-high-high- throughputthroughputthroughputthroughput

analysisanalysisanalysisanalysis ofofofof naturalnaturalnaturalnatural productsproductsproductsproductsJieJieJieJie MengMengMengMeng1111,,,, ShihuaShihuaShihuaShihua WuWuWuWu1111

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

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

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.

A

B

II

III



P-16P-16P-16P-16GentleGentleGentleGentle andandandand high-yieldhigh-yieldhigh-yieldhigh-yield preparativepreparativepreparativepreparative separationseparationseparationseparation methodmethodmethodmethod ofofofof basophilsbasophilsbasophilsbasophils usingusingusingusing aaaa

rotary-seal-freerotary-seal-freerotary-seal-freerotary-seal-free continuous-flowcontinuous-flowcontinuous-flowcontinuous-flow centrifugecentrifugecentrifugecentrifugeHiroyuki Shiono1*, Takuya Matsui1, Tadashi Okada1, Hong Miao Chen2, and Yoichiro Ito3

1Department of Physiology, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan2Huaqiao 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:Keywords:Keywords: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 consideredas primary effector cells in allergic disorders such as anaphylaxis, asthma, hay fever, atopic dermatitis (AD) andurticaria. However, the studies on basophils have been hampered by their small count in peripheral blood and thelack of appropriate animal model.

Since the novel role of basophils on chronic allergic inflammation in an animal model has been reported[1, 2], andmethods for the purification of basophils with the specific antibody have been develoed[3], studies on basophilsrecently attracted much attention. However, the negatively selecting magnetic cell separation method for obtainingpure 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 ofdepleting erythrocytes and harvesting leukocytes.

Therefore, we have developed a preparative separationl method of basophils using a rotary-seal-free continuous-flowcentrifuge 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 tohematocrit 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 CD203cantigen, the activation marker of basophils, was measured using flow cytometry before and after activation withanti-IgE.

The average concentration of basophils from healthy volunteers was 23% in the density of 1.075 and 64% in thedensity of 1.080 in three experiments. The total yield of basophils was approximately 3.6 x 105 cells from 10 ml ofthe peripheral blood. Basophils from an AD patient was also concentrated in the density of 1.075 at 22% and in thedensity 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 withanti-IgE, the increase of CD203c level in healthy volunteers and the AD patient were found in 51% and 81% ofbasophils, which were similar to the native basophils in whole blood, respectively. The separated basophils preservedIgE receptors.

These results suggested that the present method could separate even the sensitive basophils from the peripheral bloodof AD patients as functionally viable basophils.

We believe that the present method is useful for a preparative and gentle separation of basophils and other functionalcells in high yield.


1. K. Obata, K. Mukai, Y. Tsujimura, K. Ishiwata, Y. Kawano, Y. Minegishi, N. Watanabe, H. Karasuyama, Blood110 (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.


P-17P-17P-17P-17BetalainBetalainBetalainBetalain separationseparationseparationseparation fromfromfromfrom redredredred beetbeetbeetbeet juicejuicejuicejuice ((((BetaBetaBetaBeta vulgarisvulgarisvulgarisvulgaris L.)L.)L.)L.) bybybyby high-performancehigh-performancehigh-performancehigh-performancecountercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography (HPCCC)(HPCCC)(HPCCC)(HPCCC) inininin polarpolarpolarpolar high-salthigh-salthigh-salthigh-salt solventsolventsolventsolvent systemssystemssystemssystems

AnetaAnetaAnetaAneta SpSpSpSpóóóórna-Kucabrna-Kucabrna-Kucabrna-Kucab1*1*1*1*,,,, SvetlanaSvetlanaSvetlanaSvetlana IgnatovaIgnatovaIgnatovaIgnatova2222,,,, IanIanIanIan GarrardGarrardGarrardGarrard2222,,,, SSSSłłłławomirawomirawomirawomir WybraniecWybraniecWybraniecWybraniec1111,,,,Cracow University of Technology, Faculty of Chemical Engineering and Technology, Institute C-1, Department

of Analytical Chemistry, Cracow, ul. Warszawska 24, Poland2Brunel 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) byconsumers has created an increased interest in red beet (Beta vulgaris L.) as a low-cost, natural source ofthese pigments (1). The application of betalains is restricted due to their low stability in certainphysicochemical conditions (e.g. oxygen, light, temperature etc.). Decarboxylated and dehydrogenatedbetalains usually are more stable than their corresponding betalains (2). The low stability of betalains generatessome problems with their isolation. So far, the separation of complex mixtures has been realized mainly byHPLC (3). However, the introduction of high-speed countercurrent chromatography (HSCCC) generated newopportunities for obtaining larger amounts of pure pigments whereby the betalains separation was performedmainly 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 thermaltreatment 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 separationof decarboxy-/dehydro-betalains was performed for the first time. The best results were obtained for mixture 1in systems I and II in which separation of 2,17-bidecarboxy-betanin, 2-decarboxy-betanin and neobetanin wasvery effective. In contrast, the separation of betanin from 17-decarboxy-betanin was less effective. Thesecompounds eluted during the elution extrusion process. The application of these solvent systems to theseparation 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.


(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.


P-18P-18P-18P-18SeparationSeparationSeparationSeparation ofofofof decarboxy-/dehydro-betalainsdecarboxy-/dehydro-betalainsdecarboxy-/dehydro-betalainsdecarboxy-/dehydro-betalains bybybyby high-performancehigh-performancehigh-performancehigh-performance

countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography inininin ion-pairion-pairion-pairion-pair solventsolventsolventsolvent systems.systems.systems.systems.Aneta Spórna-Kucab1*, Svetlana Ignatova2, Ian Garrard2, Sławomir Wybraniec1

Cracow University of Technology, Faculty of Chemical Engineering and Technology, Institute C-1, Department ofAnalytical Chemistry, Cracow, ul. Warszawska 24, Poland

2 Brunel University, Brunel Institute for Bioengineering, Uxbridge, West London, UK*[email protected]

Keywords:Keywords:Keywords: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 betacyaninis 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-toxicfeatures, so they are very attractive compounds for consumers and scientists. Research on the different structures ispossible 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 acylatedderivatives (4).

In this study, ion-pair high-performance countercurrent chromatography (HPCCC) was used for the first time toseparate 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 HPCCCprocess 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 – theion-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 higherconcentrations. The application of the ion-pair solvent systems resulted in betalain chromatographic profiles thatwere significantly different from the profiles observed in C18-HPLC, due to the formation of ion-pairs between thenon-dehydrogenated betalains and anions of the acids. The study confirmed that the ion-pair solvent system withHFBA 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 forthe more polar pigments in the mixture 1 (betanin, 17-decarboxy-betanin, 2-decarboxy-betanin,2,17-bidecarboxy-betanin, and neobetanin).

References:References:References: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 inreversed-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 polarbetalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography. J. Chromatogr. A2009, 1216, 6890-6899.


P-19P-19P-19P-19DevalopmentDevalopmentDevalopmentDevalopment ofofofof newnewnewnew patternpatternpatternpattern PTFEPTFEPTFEPTFE columncolumncolumncolumn ofofofof HSCCCHSCCCHSCCCHSCCC

WangWangWangWang Gao-hongGao-hongGao-hongGao-hong1,21,21,21,2,,,, ChenChenChenChen Xiao-FenXiao-FenXiao-FenXiao-Fen1,21,21,21,2,,,, HuangHuangHuangHuang Xin-YiXin-YiXin-YiXin-Yi1111,,,, YunYunYunYun LiuLiuLiuLiu1,21,21,21,2,,,, DiDiDiDi Duo-LongDuo-LongDuo-LongDuo-Long1111****1Key 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:Keywords:Keywords:Keywords: Column; PTFE; Flavonoid; HSCCCBased on the multilayer coil polytetrafluroethylene (PTFE) column as separation column, the separation andpurification of chemical components from complex matrix was carryed out using suitable two-phase solvent systemon HSCCC. The separation mechanism of conventional HSCCC relegates to a continuous liquid–liquid partitionchromatography 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 complexsamples depended simply on single liquid–liquid partition model. In order to improve separation efficiency onHSCCC to compounds which possess similar structure and distribution coefficient in complex samples, a new patternof PTFE column would be developed.

Firstly, in order to increase inner-surface roughness and enlarge surface area, sodium naphthalene solution wasemployed for pre-treating the inner-surface of PTFE column. Secondly, PTFE column was filled with dopaminesolution and the dopamine self-assembly was performanced on the inner-surface of PTFE column. The modifiedprocess is shown in figure 1. As observed in SEM graphs (figure 2), the inner-surface of untreated PTFE column wasquite smooth (see figure 2-a), while that of treated PTFE column was rather rough (see figure2-b). Poly-dopamineparticles could be found on the inner-surface of coated PTFE column (see figure 2-c). The treated PTFE column wasused for separation of three flavonoid compounds (quercitin, kaempferol and isorhamnetin). The result showed thatthis new pattern PTFE column could visibly improve separation efficiency of three flavonoid compoundsabove-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:Acknowledgements:Acknowledgements: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).



P-20P-20P-20P-20MMMMultipleultipleultipleultiple dualdualdualdual modemodemodemode centrifugalcentrifugalcentrifugalcentrifugal partitionpartitionpartitionpartition chromatographychromatographychromatographychromatography asasasas anananan efficientefficientefficientefficientmethodmethodmethodmethod forforforfor thethethethe purificationpurificationpurificationpurification ofofofof 4-phenylcoumarins4-phenylcoumarins4-phenylcoumarins4-phenylcoumarins fromfromfromfrom MMMMesuaesuaesuaesua eleganseleganseleganselegans....

ChanChanChanChan GomathiGomathiGomathiGomathi1111,,,, JoelJoelJoelJoel BoustieBoustieBoustieBoustie2222,,,, GrGrGrGréééégoiregoiregoiregoire Audo*Audo*Audo*Audo*3333,,,, NorNorNorNor HadianiHadianiHadianiHadiani IsmailIsmailIsmailIsmail4444,,,, CCCCéééélinelinelineline LeLeLeLe QuQuQuQuéééémenermenermenermener3333,,,,KhalijahKhalijahKhalijahKhalijah AwangAwangAwangAwang1111

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, France3Armen instrument, ZI Kermelin, 16 rue Ampère, 56890 Saint Avé, France.

[email protected] 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 Mesuaelegans (Clusiaceae). A direct comparison between classical isocratic mode and multi-dual mode with atwo-phase solvent system composed of heptane-ethyl acetate-acetonitrile-water (9:1:9:1, v/v/v/v) wasperformed. 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), wereobtained from the extract with 280 nm HPLC purity of 93.2 % and 94.3 % in 500 mg injection in multi-dualmode (Table 1), compare to 82.3% and 79.4% in isocratic mode (Table 2). Structures of these two compoundswere identified by 1H NMR, 13C NMR and LC-MS. Multi-dual mode allows in these case to increase theinjection capacity without decreasing purity of target compounds.

1 2

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

Multi-dualMulti-dualMulti-dualMulti-dual modemodemodemode

Mass

InjectedMammea A/BA1 Mesuagenin C2

50 mg 99.7% 100%

150 mg 97.6% 95.5%

300 mg 93.0% 96.6%

500 mg 93.2% 94.3%

Table 1. 280 nm HPLC purity of Mammea A/BAand Mesuagenin C after multi dual mode CPC

IsocraticIsocraticIsocraticIsocratic descendingdescendingdescendingdescending modemodemodemode

Mass

InjectedMammea A/BA1 Mesuagenin C2

50 mg 98.5% 98.1%

150 mg 96.2% 94.6%

300 mg 84.2% 91.1%

500 mg 82.3% 79.4%

Table 2. 280 nm HPLC purity of Mammea A/BA andMesuagenin C after isocratic CPC mode.



P-21P-21P-21P-21PPPPreparativereparativereparativereparative separationseparationseparationseparation ofofofof twotwotwotwo subsidiarysubsidiarysubsidiarysubsidiary colorscolorscolorscolors ofofofof FD&CFD&CFD&CFD&C yellowyellowyellowyellow NO.NO.NO.NO. 5555

(tartrazine)(tartrazine)(tartrazine)(tartrazine) bybybyby spiralspiralspiralspiral high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyAdrianAdrianAdrianAdrian WeiszWeiszWeiszWeisza*a*a*a*,,,, JoseJoseJoseJose A.A.A.A. RoqueRoqueRoqueRoqueaaaa,,,, EugeneEugeneEugeneEugene P.P.P.P. MazzolaMazzolaMazzolaMazzolabbbb,,,, YoichiroYoichiroYoichiroYoichiro ItoItoItoItocccc

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:Keywords: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, andcosmetics in the U.S. (1). It is manufactured by condensing phenylhydrazine-p-sulfonic acid with an oxalaceticester, coupling the product with diazotized sulfanilic acid, and then hydrolyzing the ester with sodiumhydroxide (2). The reaction produces mainly 1111 (Fig. 1, azo form) accompanied by a variety of organicimpurities, which include intermediates, side-reaction products, and subsidiary colors. These impurities arecarried

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 FederalRegulations (CFR). Among the

specifications is a limit of 1% for each of two polysulfonated subsidiary colors labeled “Pk5”

and “Pk7” in Figure 2.

Figure 2 HPLC chromatogram of the sample of Y5 usedin the present work.

Currently, the determination of these subsidiary colors is performed by an indirect method because FDA doesnot have reference materials for Pk5 and Pk7. The present work describes the use of spiral high speedcounter-current chromatography (HSCCC) to separate preparative quantities of Pk5 and Pk7 from a sample ofY5 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 instrumentused was a CCC-1000 Pharma-Tech J-type HSCCC fitted with three spiral-tube assembly coils (CC BiotechLLC) resulting in a column with total capacity of 225 ml. The solvent system 1-butanol-ethanol-saturatedammonium sulfate-water (1.6:0.4:1:1, v/v) was chosen based on the graphic optimization of the partitioncoefficients 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 separatedby using the organic phase as the mobile phase. The separated components were identified by NMR andhigh-resolution MS techniques. The separated materials will be used for qualitative and quantitativeconfirmatory analyses in order to enforce the CFR specifications for Pk5 and Pk7 in Y5.ReferencesReferencesReferencesReferences1. 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.

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



P-22P-22P-22P-22NovelNovelNovelNovel samplesamplesamplesample pretreatmentpretreatmentpretreatmentpretreatment methodmethodmethodmethod forforforfor increasingincreasingincreasingincreasing thethethethe preparativepreparativepreparativepreparative scalescalescalescale ofofofof

pH-zonepH-zonepH-zonepH-zone refiningrefiningrefiningrefining counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyYiYiYiYi Yang,Yang,Yang,Yang, DongyuDongyuDongyuDongyu Gu,Gu,Gu,Gu, QibienQibienQibienQibien Chen,Chen,Chen,Chen, AbulimitiAbulimitiAbulimitiAbulimiti Yili,Yili,Yili,Yili, HajiHajiHajiHaji AkberAkberAkberAkber Aisa*Aisa*Aisa*Aisa*

Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute ofPhysics 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:Keywords:Keywords:Keywords: crude sample preparation, yield, pH-zone refining counter-current chromatography

pH-zone refining counter-current chromatography (CCC) is classic large-scale preparative technique forseparating ionizable compounds [1,2]. The typical sample size separated by high-speed counter-currentchromatography (HSCCC) instrument with 320 ml column capacity may range from 0.5 to 5 g [3]. As weknown, the selection of solvent system is the most important step for pH-zone-refining CCC separation [3]. Foran acidic analyte, when pH of two-phase solvent system is around 2, Kacid should be much bigger than 1. Andfor a basic analyte, when pH of solvent system is about10-12, Kbase shold be much bigger than 1 [3]. Usually, afterselection of two-phase solvent system, pH-zone-refiningCCC sparation procedure will begin imediataly. However,if the selected two-phase solvent system without acid orbase in the lower phase is used for extraction of targetcompound, most of this compound would be enriched inthe upper phase because of very large K value of targercompound in this solvent system. So the content of targetcompound would be increased. If the same crude samplemount was loaded into CCC column, the yeild of targetcompound will increased. Base on this idea, a novelmethod for crude sample pretreatment forpH-zone-refining CCC sparation was established in thepresent study. The separation of alkaloids with antitumoractivity in the seeds of Sophora alopecuroides, atraditional medicine, was presented as example of ourmethod [4]. Two-phase solvent system composed ofmethyl tert-butyl ether-acetonitrile-water (2:2:3, v/v),where triethylamine (10mM) was added to the organicphase as a retainer and hydrochloric acid (10 mM) to theaqueous phase as an eluter, was selected and used in theseparation of pH-zone-refining CCC. Before CCCseparation, 20 g total alkaloids (crude sample I) wasextrated by this solvent system, where triethylamine (10mM) was added to the organic phase. And 10 g crudesample II was obtained. 2 g crude sample I and II were separated by pH-zone-refining CCC under the sameconditions, respectively. And 98 mg sophoramin and 371 mg

sophoearpin were isolated from crude sample I (Fig. 1A). And 169 mg sophoramin and 696 mg sophoearpinwere isolated from crude sample II (Fig. 1B). The purities of products were similar and over 95%. And whenthe 1 g crude sample II from 2 g crude sample I was separated by CCC. The retention of stationary phase wasremarkable increased. And similar mounts of two compounds were obtained. Thus, the target compounds wereefficiently enriched by this sample pretreatment method in the present study. Although one extraction step wasadded in the procedure of crude sample preparation, the yields of products of pH-zone-refining CCC wereincreased to approximately 2 times.


1. Weisz, A.; Scher, A. L.; Shinomiya, K.; Fales, H. M.; Ito, Y. J. Am. Chem. Soc. 1994199419941994, 116, 704-708.2. Ito, Y; Ma, Y. J. Chromatogr. A 1996199619961996, 753, 1-36.3. Ito, Y. J. Chromatogr. A 2005200520052005, 1065, 145-168.4. Zhang L; Wang T; Wen X; Wei Y; Peng X; Li H, Wei L. Eur. J. Pharmacol. 2007200720072007, 563, 69-76.

Figure 1. Chromatograms of alkaloids separationfrom crude sample I (A) and II (B) of Sophoraalopecuroides seeds obtained by pH-zonerefining CCC.


P-23P-23P-23P-23SSSSolventolventolventolvent gradientgradientgradientgradient counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography purificationpurificationpurificationpurification ofofofof

podophyllotoxinpodophyllotoxinpodophyllotoxinpodophyllotoxinssss fromfromfromfrom DDDDysosmaysosmaysosmaysosma versipellisversipellisversipellisversipellis (hance)(hance)(hance)(hance)ZhiZhiZhiZhi YangYangYangYang1111,,,, ShihuaShihuaShihuaShihua WuWuWuWu1111****

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:Keywords:Keywords: solvent gradient counter-current chromatography; linear solvent gradient ccc separation;step-gradient ccc separation; podophyllotoxins;

Abstract:Abstract:Abstract:Abstract: Stepwise elution modes have been frequently applied in the CCC separation to isolate compounds with alarge range of polarities. During the preparation of Dysosma versipellis (Hance) by HSCCC, all the compounds canbe 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. Inhexane-ethyl acetate-methanol-water (4637) system, compounds 1 and 2 can be separated much better, while it takesa much longer time. When a stepwise elution mode is applied, sound separation and short time are obtained. In thiswork we applied two stepwise elution modes--Linear solvent gradient CCC separation and Step-gradient CCCseparation. And Step-gradient CCC separation proved more effective in this work. In linear solvent gradient CCCseparation, the changes of the system are more mitigatory and the reduction of the stationary phase exists during thewhole changing process. While in the step-gradient CCC separation, new hydrodynamic equilibrium can beestablished 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 moreeffective mode should be selected.

Figure 1.Stuctures of podophyllotocins

separated from dysosma versipellis (hance)

Figure2.The elution curves of the preparative CCC. 1111. 4′-demethylpodophyllotoxin 2222. β-peltatin-5-O-β-D-glucopyranoside 3333.β-peltatin 4444. podophyllotoxin. CCC separation conditions: A: Stationary phase, the upper phase of HEMWat (4646). Mobilephase, the lower phase of HEMWat (4646), Retention of the stationary phase, 59.26%. B: A Stationary phase, the upper phase ofHEMWat (4637). Mobile phase, the lower phase of HEMWat (4637), Retention of the stationary phase, 61.11%. C: Stationaryphase, the upper phase of HEMWat (4637). Mobile phase, 0-120 min, the lower phase of HEMWat (4637), 120-255 min, thelower phase of HEMWat (4637) from 100% to 0 and the lower phase of HEMWat (4646) from 0 to 100%, after 255 min , thelower phase of HEMWat (4646). D: Stationary phase, the upper phase of HEMWat (4637). Mobile phase, 0-120 min, the lowerphase 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 theupper and lower phases (1:1, v/v, 4 mL of the total volume)..

O

OO

O

R2 R3

MeO

R1

OMe

1'2'

3'4'5'

6'

12a2

3a34 10 5

6

789

R1 R2 R34′-demethylpodophyllotoxin (1111): -OH -OH -H β-peltatin-Glu (2222): -OCH3 -H -Glu β-peltatin (3333): -OCH3 -H -OH podophyllotoxin (4444): -OCH3 -OH -H


http://dict.bioon.com/detail.asp?id=a6ee559983


P-24P-24P-24P-24PPPPreparativereparativereparativereparative separationseparationseparationseparation ofofofof isoquiinolineisoquiinolineisoquiinolineisoquiinoline alkaloidsalkaloidsalkaloidsalkaloids fromfromfromfrom coptiscoptiscoptiscoptis japonicajaponicajaponicajaponica bybybyby

high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyGuijaeGuijaeGuijaeGuijae YooYooYooYooaaaa,,,, TaeTaeTaeTae BumBumBumBum KimKimKimKimaaaa,,,, HeejungHeejungHeejungHeejung YangYangYangYangaaaa,,,, Jin-HoJin-HoJin-HoJin-Ho parkparkparkparkbbbb,,,, YoungYoungYoungYoung ChoongChoongChoongChoong KimKimKimKima,ba,ba,ba,b,,,,

SangSangSangSang HyunHyunHyunHyun SungSungSungSunga,b,*a,b,*a,b,*a,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 ofKorea

****Corresponding author: [email protected]

Keywords:Keywords:Keywords: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 extractof Coptis japonica Makino (Ranunculaceae). The two phase solvent system comprisingchloroform–methanol–0.2 M hydrochloric acid (4:1.5:2, v/v/v) was employed to HSCCC and the flow rate ofmobile phase was optimized at 1.5 ml/min and rotated at 800 rpm. The monitoring of HSCCC peak fractionswas performed by combining effluent line of the HSCCC apparatus to UV detector (280 nm). Isolatedcompounds were elucidated by ESI-MS and several NMR techniques including 1D and 2D NMR spectroscopicmethods.

#### fractionfractionfractionfraction CompoundCompoundCompoundCompound ParentParentParentParent ionionionion([M]([M]([M]([M]++++))))

YieldYieldYieldYield(mg)(mg)(mg)(mg)

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

Figure 1. HSCCC chromatography of the Coptisjaponica n-BuOH extracts

Figure 2. HPLC-ESI-MS spectrum of the five isoquinolinealkaloids in Coptis japonica n-BuOH extracts

CompoundCompoundCompoundCompound RRRR1111 RRRR2222 RRRR3333 RRRR4444

Palmatine CH3 CH3 CH3 CH3

Berberine CH3 CH3 —CH2—

Epiberberine —CH2— CH3 CH3

Coptisine —CH2— —CH2—

Columbamine CH3 CH3 CH3 H

Figure 3. The chemical structures of isoquinoline alkaloids in then-BuOH extract of Coptis japonica


1. Yang, F; Zhang, T; Zhang, R; Ito, Y. J. Chromatogr. A.1998199819981998, 829, 137.2. Yoon, K. D.; Chin, Y. W.; Yang, M. H.; Kim, J. W.Food Chem. 2011201120112011, 129, 679.


P-25P-25P-25P-25ScreeningScreeningScreeningScreening forforforfor anti-diabetesanti-diabetesanti-diabetesanti-diabetes fractionfractionfractionfraction inininin thethethethe leavesleavesleavesleaves ofofofof

OOOOlealealealea europaeaeuropaeaeuropaeaeuropaea LLLL.... bybybyby HPCCCHPCCCHPCCCHPCCCZhangZhangZhangZhang JiaJiaJiaJia1,31,31,31,3,,,, HuangHuangHuangHuang Xin-YiXin-YiXin-YiXin-Yi1111,,,, PeiPeiPeiPei DongDongDongDong1111,,,, ChenChenChenChen Xiao-FenXiao-FenXiao-FenXiao-Fen1,21,21,21,2,,,, DiDiDiDi Duo-LongDuo-LongDuo-LongDuo-Long1,31,31,31,3****

1 Key Laboratory of Chemistry of Northwestern Plant Resources & Key Laboratory for Natural Medicine of GansuProvince, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000

2 Graduate University of the Chinese Academy of Sciences, Beijing 1000493Department of Pharmacy，Gansu College of Traditional Chinese Medicine，Lanzhou 730000

*Corresponding author fax: 0931-4968094; e-mail: [email protected]

Keywords:Keywords:Keywords:Keywords:Olea europaea L; anti-diabete; HPCCCModern 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 reportsabout 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 extractsof the Olea europaea L. leaves. The α-amylase and non-enzymatic glycation system were employed to evaluate theanti-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 performancedby HPCCC (High Performance Counter-Current Chromatography).

The separation was achieved on a DE Spectrum HPCCC under analytical condition with solvent system ethylacetate–water (1:1, v/v). The upper phase as stationary phase and mobile phase was pumped at a flow rate of 0.8mL/min at 1500 rpm. Six constituents were obtained (See figure 1), and the anti-diabetes activity was evaluatedusing above mentioned method. The result was found that four constituents were screened out to possess potentialactivities.

Owing to the numbered 1 constituent in figure 1 was admixture , further separation was performanced asfollows: butanol–water-acetic acid (1:1:0.1, v/v) was selected as solvent system, the upper phase was stationary phaseand 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 possesspotential activities.

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

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



P-26P-26P-26P-26FlowFlowFlowFlow separationseparationseparationseparation ofofofof cellscellscellscells inininin counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography (CCC)(CCC)(CCC)(CCC) centrifugescentrifugescentrifugescentrifuges

TianTianTianTian Han,Han,Han,Han, RemcoRemcoRemcoRemco vanvanvanvan dendendenden Heuvel,Heuvel,Heuvel,Heuvel, IanIanIanIan AAAA SutherlandSutherlandSutherlandSutherland andandandand DerekDerekDerekDerek FisherFisherFisherFisherAdvanced 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:Keywords:Keywords:Keywords: Flow separation, cell separation, CCC centrifuge

Stem cell therapy requires the separation of differentiated from undifferentiated stem cells. The aim of ourresearch is to investigate and develop new cell separation methods based on flow separation in counter-currentchromatog-raphy (CCC) centrifuges that could be applied to stem cell separations. There have been few reports ofcell separations by CCC. Ito et al separated red blood cells (RBCs) using a non synchronous CCC centrifuge in asingle 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 flowrate 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 synchronousCCC centrifuge: a Milli-CCC J-type centrifuge (3) with an 8.2 mL, 1 mm ID, multilayer column, filled with anisotonic NaCl-phosphate buffer (pH 7.4) (PBS). RBCs in saline were injected and 1ml fractions collected. Inlinemonitoring at 405nm detected cells and haemoglobin (Hb). Free Hb and intact cells containing Hb weredistinguished 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 inthe column volume. For a particular rotational speed there was a minimum flow rate which caused all the cells tobe retained and a maximum flow rate when all cells were eluted. Between these two flow rates some cells wereretained and some eluted. When the flow rate was increased from the minimum flow rate retained cells could beeluted from the column (Fig 1).

Human RBCs had a higher min flow rate than the sheep RBCs, indicating that human RBCs are retainedeasier than sheep RBCs. But, human RBCs also have a lower max flow rate, indicating that human RBCs are alsopushed 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) usinga nonsynchronous CCC indicating a difference in the separation mechanisms.

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 flowto 5ml/min (after arrow point) eluted the RBCs (Peak B). A405 before and after centrifugation distinguishes intact cells from free Hb.

Fig.2Cell separation. A mixture of sheep and human RBCs was retained in the coil at 800rpm, 0.5ml/min and then eluteddifferentially 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 coilwill also be reported.

ReferencesReferencesReferencesReferences1.Ito Y, Carmeci P & Sutherland, IA (1979) Anal. Biochem. 94, 249-2522. Sutherland, IA & Ito Y (1980) Anal. Biochem. 108, 367-3733. Janaway L, Hawes D, Ignatova S, Wood P & Sutherland IA (2003) J. Liq. Chrom.R.T., 26, 9-10, 1345-1354.

A

Human RBCs

Sheep RBCs

B


P-2P-2P-2P-27777ComparativeComparativeComparativeComparative studystudystudystudy ofofofof threethreethreethree modificationsmodificationsmodificationsmodifications ofofofof thethethethe controlled-cyclecontrolled-cyclecontrolled-cyclecontrolled-cycle liquid-liquidliquid-liquidliquid-liquidliquid-liquid

chromatographychromatographychromatographychromatography devicedevicedevicedeviceAndreyAndreyAndreyAndrey A.A.A.A. Erastov,Erastov,Erastov,Erastov, YulyaYulyaYulyaYulya A.A.A.A. Zakhodjaeva,Zakhodjaeva,Zakhodjaeva,Zakhodjaeva, AndreyAndreyAndreyAndrey A.A.A.A. Voshkin,Voshkin,Voshkin,Voshkin,

ArtakArtakArtakArtak E.E.E.E. KostanyanKostanyanKostanyanKostanyan****

Kurnakov Institute of General & Inorganic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 31, Moscow119991, Russia fax +495 9554834, e-mail: [email protected]

Keywords:Keywords:Keywords: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 andcoumarin. The CLC apparatus consists of a series of multistage columns connected in the form of a coil, and apulsation-cycling flow system for regulated discrete supply of the mobile phase as repetitive impulses to theapparatus (1,2). The columns are divided into stages by equally spaced horizontal perforated plates. Thepulsation-cycling flow system presents a piston pump with adjustable piston stroke length and PC regulatedfrequency and piston velocity. An operating cycle of the CLC apparatus consists of two individually timedperiods: 1 - flow (contact) period, during which a specified portion of mobile phase is displaced downwardbeing dispersed in the stationary phase in each stage of the columns; 2 - settling period, during which thedroplets 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 (fromPTFE sheet, 3 mm thick with 0.25 mm diameter holes) at 35 mm intervals; 2 – column of 8 mm internaldiameter 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 PTFEperforated plates (2 mm thick with 0.25 mm diameter holes) at 15 mm intervals. Column 3 is equipped withagitators, located in each stage and operating in the cyclic (periodic) mode. The chromatographic peaks ofcaffeine (KD =0.58) and coumarin (KD =1.65) obtained by using column 3 with the hexane-isopropanol-waterbiphasic 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 (withagitators).

The advantage of the column3 (with agitators) over the first two versions is the possibility to adjust the contacttime for each specific separation process. This is particularly important when the interphase mass transfer isaccompanied by chemical reactions, as for example, in the processes of metal separation. Of all threeinvestigated columns, by using hexane-isopropanol-water biphasic system column 3 demonstrated the highestefficiency (measured with the number of theoretical plates).


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.


P-28P-28P-28P-28TargetedTargetedTargetedTargeted analysisanalysisanalysisanalysis ofofofof naturalnaturalnaturalnatural productsproductsproductsproducts bybybyby K-basedK-basedK-basedK-based countercurrentcountercurrentcountercurrentcountercurrent separationseparationseparationseparationFengFengFengFeng QiuQiuQiuQiu1111,,,, J.J.J.J. BrentBrentBrentBrent FriesenFriesenFriesenFriesen1,21,21,21,2,,,, JamesJamesJamesJames B.B.B.B. McAlpineMcAlpineMcAlpineMcAlpine1111,,,, DavidDavidDavidDavid C.C.C.C. LankinLankinLankinLankin1111,,,, andandandand GuidoGuidoGuidoGuido F.F.F.F.

PauliPauliPauliPauli1111,,,,****

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 distributedbetween a liquid stationary phase and a liquid mobile phase. The K value is not only an important theoreticalparameter in CS, but also has much practical value. In this study, K values were used to predict the CS profilesof analytes, assist in the selection and optimization of CS solvent systems, and guide targeted separation. Thepresent study developed a quantitative 1H NMR (qHNMR)-based approach for the simultaneous measurementof 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 toenhance the resolution of these difficult-to-separate natural product congeners. Using the K values in optimizedorthogonal solvent systems, a “K map” was created that guided locating the target compounds in series of CSfractions (see Figure 1). Consequently, all five terpene lactones could be separated, yielding isolates withqHNMR purities of > 95%, directly from Ginkgo biloba leaf extracts.

The separation was further evaluated by offline qHNMR analysis of CS fractions (offline CS-NMR) associatedwith Gaussian curve fitting. The Gaussian nature of CS elution profiles results from CS theory and is supportedby the experimental NMR-hyphenated CS data. NMR-guided and targeted K-based countercurrent separationwas 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 highlyversatile platform for the targeted analysis of natural products and other complex materials, utilizing thepartition 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 CSin a pair of specially designed SSs.


P-2P-2P-2P-29999CharacterisationCharacterisationCharacterisationCharacterisation ofofofof quaternaryquaternaryquaternaryquaternary HeptaneHeptaneHeptaneHeptane----WaterWaterWaterWater basedbasedbasedbased phasephasephasephase systemssystemssystemssystems acrossacrossacrossacross thethethethe

polaritypolaritypolaritypolarity rangerangerangerange forforforfor Counter-currentCounter-currentCounter-currentCounter-current ChromatographyChromatographyChromatographyChromatographyR.N.A.M.R.N.A.M.R.N.A.M.R.N.A.M. vanvanvanvan dendendenden HeuvelHeuvelHeuvelHeuvel1111,,,, G.G.G.G. HarrisHarrisHarrisHarris2222,,,, N.N.N.N. DouilletDouilletDouilletDouillet3333,,,, C.C.C.C. ThickittThickittThickittThickitt3333,,,,

E.A.E.A.E.A.E.A. vanvanvanvan dendendenden HeuvelHeuvelHeuvelHeuvel1111,,,, S.S.S.S. IgnatovaIgnatovaIgnatovaIgnatova1,*1,*1,*1,*


2 Dynamic Extractions Inc, NJ 08852, USA3GlaxoSmithKline, Pharmaceuticals R&D Facility, Stevenage, SG1 2NY, UK

Keywords:Keywords:Keywords: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 thistechnique. A CCC column simply consists of a biphasic mixture of liquids with the separation being determinedby the solute distribution ratio between the two phases. The number of potential columns (solvent systems) isalmost infinite. However, solvent system selection, equivalent to column selection for HPLC and SFC, stillremains 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 demonstratedallowing the automation of the solvent system scouting process (3,4). This has enabled rapid development ofseparations for solutes compatible with the HEMWat series. If a sample is not HEMWat compatible there arelimited examples of alternative solvent system tables.

To date there are at least two approaches described in the literature for developing solvent system tables. In oneapproach, demonstrated by HEMWat, the overall polarity difference between phases remains the same. Thistype of systems covers a wide range of polarity. Another approach, for example a Heptane, Toluene, Acetoneand 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 HTAWsystem 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 andsolubility for small molecule separations. Both solvent system development approaches were applied toHeptane-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 testedusing a model mixture (6).

Figure 1 shows how the selectivity for the seven components in the mixture is very similar for the HTAW andHEMWat 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 phasesystems.

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsThe TSB-STEP team would like to acknowledge financial support from the Technology Strategy Board’s HighValue Manufacturing programme (Grant No. TSB-HVM/BD506K).ReferencesReferencesReferencesReferences1. Berthod, A.; Hassoun, M.; Ruiz-Angel, M.J. Anal. Bioanal. Chem. 2005,2005,2005,2005, 383, 327-340.2. Garrard, I.J. J Liq. Chrom. & Rel. Technol. 2005,2005,2005,2005, 28, 1923-1935.3. Wu, D.; Jiang, X.; Wu, S. J. Sep. Sci. 2012,2012,2012,2012, 33, 67-73.4. Garrard, I.J.; Janaway, L.; Fisher, D. J. Liq. Chrom. & Rel. Technol. 2007,2007,2007,2007, 20, 151-163.5. Renault, J.H.; Nuzillard, J.M.; Intes, O.; Maciuk, A. In: Countercurrent chromatography, the support-free liquid stationary phase (Comprehensiveanalytical chemistry, vol. 38) Berthod A (ed) Elsevier, Amsterdam, 2002200220022002; pp 49-83.6. Ignatova, S.; Sumner, N.; Colclough, N.; Sutherland, I. J. Chrom. A. 2011201120112011, 1218, 6053-6060.


P-P-P-P-30303030Large-ScaleLarge-ScaleLarge-ScaleLarge-Scale PreparativePreparativePreparativePreparative ProfilingProfilingProfilingProfiling ofofofof AnacardicAnacardicAnacardicAnacardic AcidsAcidsAcidsAcids inininin CashewCashewCashewCashew NutNutNutNut ShellShellShellShell OilOilOilOil

LiquidLiquidLiquidLiquid bybybyby Spiral-CoilSpiral-CoilSpiral-CoilSpiral-Coil CountercurrentCountercurrentCountercurrentCountercurrent ChromatographyChromatographyChromatographyChromatography andandandand Off-LineOff-LineOff-LineOff-LineESI-MS-MSESI-MS-MSESI-MS-MSESI-MS-MS ContinuousContinuousContinuousContinuous InfusionInfusionInfusionInfusion

MarianaMarianaMarianaMariana NevesNevesNevesNeves VieiraVieiraVieiraVieira 1111 ,,,, KathrinKathrinKathrinKathrin WelkeWelkeWelkeWelke 1111 ,,,, JosuJosuJosuJosuéééé A.A.A.A. Murillo-VelMurillo-VelMurillo-VelMurillo-Veláááásquezsquezsquezsquez 2222 ,,,,SuzanaSuzanaSuzanaSuzana GuimGuimGuimGuimããããresresresres LeitLeitLeitLeitããããoooo 3333 ,,,, PeterPeterPeterPeter WinterhalterWinterhalterWinterhalterWinterhalter ****,1,1,1,1 ,,,, GeroldGeroldGeroldGerold JerzJerzJerzJerz 1111

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 Salvador3 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, thecashew 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 biologicalactivities 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 (Leishmaniasp.) (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 HCTion trap-MS). This method yielded a reconstituted metabolite profile and finally enabled a MS-target-guidedisolation procedure (Figure 1). Prior to the off-line infusion the fractions were diluted by the factor 50. Elutionorders and co-elution effects of minor and major concentrated anacardic acids were specifically monitored byselected ion-traces (neg. mode, scan: m/z 50-1500, [M-H] - signals) (8). The use of the spiral coil-CCC enabled thespecific recovery of homologue anacardic acids in the gram range and will be of great value for biologicalevaluations. Minor constituents were significantly enriched for later 1D/2D-NMR spectroscopic analysis.

Figure 1.ESI-MS selected anacardic acid ion traces from the continuous off-line infusion of spiralCCC-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. 1993199319931993, 41, 1012-1015.2. Muroi et al., Biosci. Biotech. Biochem. 1994199419941994, 58, 1925-1926.3. Kubo et al. J. Agric. Food Chem. 2003200320032003, 51, 7624-7628.4. Castillo-Juárez et al., J. of Ethnopharmacol. 2007200720072007, 114, 72–77.5. Lee et al., Parasitol. Res. 2009200920092009, 104, 463–466.6. Pereira et al., Bioorg. Med. Chem. 2008200820082008, 16, 8889-8895.7. Franca et al, Rev. Soc. Bras. Med. Trop. 1996199619961996, 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, 2012201220122012.

C O O H

OH R

1 7

1 0

1 7

1 7

5 5 5 5 : m /z 3 6 9

1 0

1 0

C 1 7 a n a c a r d ic a c id sC 1 7 a n a c a r d ic a c id sC 1 7 a n a c a r d ic a c id sC 1 7 a n a c a r d ic a c id sR = :R = :R = :R = :

6 6 6 6 : m /z 3 7 1

7 7 7 7 : m /z 3 7 3

8

1 1

8

1 1

1 4

81 5

1 5

1 5

R = :R = :R = :R = :

4 4 4 4 : m /z 3 4 7

3 3 3 3 : m /z 3 4 5

2 2 2 2 : m /z 3 4 3

1 1 1 1 : m /z 3 4 1

C 1 5 a n a c a r d ic a c id sC 1 5 a n a c a r d ic a c id sC 1 5 a n a c a r d ic a c id sC 1 5 a n a c a r d ic a c id s



P-P-P-P-31313131

PreparativePreparativePreparativePreparative MetaboliteMetaboliteMetaboliteMetabolite ProfilingProfilingProfilingProfiling ofofofof thethethethe DiatomDiatomDiatomDiatom MicroalgaMicroalgaMicroalgaMicroalga PhaeodactylumPhaeodactylumPhaeodactylumPhaeodactylumtricornutumtricornutumtricornutumtricornutum bybybyby High-SpeedHigh-SpeedHigh-SpeedHigh-Speed CountercurrentCountercurrentCountercurrentCountercurrent ChromatographyChromatographyChromatographyChromatography andandandand ContinuousContinuousContinuousContinuous

APCI-MS-MSAPCI-MS-MSAPCI-MS-MSAPCI-MS-MS InfusionInfusionInfusionInfusion

BeateBeateBeateBeate BonschBonschBonschBonsch 1111,,,, RobertRobertRobertRobert DillschneiderDillschneiderDillschneiderDillschneider 2222,,,, RosaRosaRosaRosa RoselloRoselloRoselloRosello 2222,,,, ClemensClemensClemensClemens PostenPostenPostenPosten 2222,,,,PeterPeterPeterPeter WinterhalterWinterhalterWinterhalterWinterhalter ****,,,, 1111,,,, GeroldGeroldGeroldGerold JerzJerzJerzJerz 1111

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-bacterialproperties. 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. againstmethicillin-resistant Staphylococcus aureus (MRSA) (1-3). Especially, in areas where conventional antibiotics areprohibited, such as in agriculture, and food preservation, or extensive use of antibiotics should be prevented, theanti-bacterial FFAs could be highly valuable and safe additives. Biofilm formation caused by bacterial adhesion inmedicinal products with resulting severe nosokomial hospital infections could possibly be reduced by surfacecoatings with FFAs. (1-3).Since almost 20 years, high-speed countercurrent chromatography (HSCCC) had been proven to be an excellentmethod for purification of natural products on larger lab-scale. P. tricornutum was cultivated in a 12L bubblephotobioreactor, and the biomass was freeze-dried. The lipophilic extract was injected to a preparative triple-coilHSCCC (PTR 1000, volume: 850 mL, mobile phase flow rate: 3.0 mL/ min, injection: 800 mg). The separation modewas ‘head-to-tail’ with the biphasic solvent system n-hexane/ acetonitrile 1:1 (v/v), and the elution- andextrusion-approach (two-column-volume method) of Berthod et al. resulted in the fractionation of the completepolarity profile of the injected sample (4). For a more specific and mass-spectrometric target-guided isolation ofbioactive metabolites, HSCCC separated fractions were monitored by a fast off-line infusion method toAPCI-MS-MS. This delivered important structural data also as basis for exact compound fractionation (5). Theoff-line infusion monitored the 6 hrs elution-extrusion HSCCC-run in a 50 min single APCI-MS-MS data file. Thisapproach largely reduced the APCI-MS-operation time in comparison to a direct on-line-HSCCC-MS couplingexperiment (5). The APCI-MS-MS system was set to monitor the most abundant 10 precursor ions with its respectiveMS/MS fragment ions. Partly, strong compound co-elution effects were perceived, but elucidated the identity of thecarotenoid fucoxanthin (1111) (6), free fatty acids (2222), hydroxy-phaeophytine (3333), diglycerides (4444), and triglycerides (5555)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]+


1. Desbois et al., JMBA 2010201020102010, 90(4), 769–774, 2. Desbois et al., Appl. Microbiol. Biotechnol. 2008200820082008, 81:755–764. 3. Desbois andSmith, Appl. Microbiol. Biotechnol. 2010201020102010, 85:1629–1642. 4. Berthod et al., Anal. Chem. 2003200320032003, 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. 2012201220122012, 166:1843–1855.



PPPP----33332222ZebrafishZebrafishZebrafishZebrafish bioassay-guidedbioassay-guidedbioassay-guidedbioassay-guided isolationisolationisolationisolation ofofofof anti-angiogenicanti-angiogenicanti-angiogenicanti-angiogenic componentscomponentscomponentscomponents bybybyby

high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography fromfromfromfrom licoricelicoricelicoricelicorice

LiwenLiwenLiwenLiwen Han,Han,Han,Han, YanqiangYanqiangYanqiangYanqiang Yuan,Yuan,Yuan,Yuan, QiuxiaQiuxiaQiuxiaQiuxia He,He,He,He, XiqiangXiqiangXiqiangXiqiang Chen,Chen,Chen,Chen, KechunKechunKechunKechun Liu*Liu*Liu*Liu*

Key Laboratory For Biosensors of Shandong Province, Biology Institute of Shandong Academy of Sciences,Jinan, 250014, China

E-mail: [email protected]

Keywords:Keywords:Keywords: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 advancedisolation technique of lead compounds of natural origin with therapeutical relevant bioassays wound be capableof enhancing work efficiency from complex multi-constituent extracts. In this study, therefore, abioassay-guided isolation strategy combined with bioactivity screening was explored to identify novel inhibitorfrom Glycyrrhiza uralensis Fisch(licorice) against aniogenesis, on the basis of zebra fish model and rapidpreparative 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 andevaluation of different extracts and compounds. Firstly, two bioactive fractions, Fr5 and Fr6 were obtainedfrom the ethyl acetate extract from licorice and 14 main compounds were found in Fr5 and Fr6 by HPLC. ThenHSCCC method was used for isolation these compounds from Fr5 and Fr6. Using the optimization ofseparation conditions, n-Hexane-ethyl acetate-methanol-water (5:6:5:5,v/v) system were successfully selectedto separate compound 1-5, and n-Hexane-ethyl acetate-methanol-water (6:5:6:5,v/v) was used for theseparation 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 singlecompounds(1-8,11-14) with purity of 90%-98% were obtained in the HSCCC separation process. Since bloodcirculation and vascular outgrowth in intersegmental vessel (ISV) in zebrafish model were found to besimultaneously 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. Theseexperimental results indicated that zebrafish bioassays combined with HSCCC may provide an alternativepathway for rapid isolation of bioactive natural products.



P-P-P-P-33333333HHHHighighighigh----speedspeedspeedspeed countercountercountercounter----currentcurrentcurrentcurrent chromatographychromatographychromatographychromatography preparativepreparativepreparativepreparative isolationisolationisolationisolation andandandand

purificationpurificationpurificationpurification ofofofof fourfourfourfour xanthonexanthonexanthonexanthone glycosidesglycosidesglycosidesglycosides fromfromfromfrom tibetantibetantibetantibetan medicinalmedicinalmedicinalmedicinal plantplantplantplant HaleniaHaleniaHaleniaHaleniaellipticaellipticaellipticaelliptica

LiLiLiLi YulinYulinYulinYulin****aaaa ,Liu,Liu,Liu,Liu YonglingYonglingYonglingYonglinga,ba,ba,ba,b,,,, WangWangWangWang PingPingPingPinga,ba,ba,ba,b,,,, ChenChenChenChen TaoTaoTaoTaoa,ba,ba,ba,b,,,, ZhaoZhaoZhaoZhao XiaoXiaoXiaoXiaoaaaa,,,,ChenChenChenChen ChenChenChenChenaaaa,,,, SunSunSunSun JingJingJingJingaaaa

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

KeyKeyKeyKey words:words:words:words: Halenia elliptica, HSCCC, xanthone glycosides

Mainly distributed in Tibet and Qinghai, Halenia elliptica has traditionally been used in Tibetan folk medicineto treat hepatitis [1]. According to prior phytochemical investigations, the principally effective components inHalenia elliptica include triperpenoids, flavonoids, and xanthones[2]. Of these components, xanthonecompounds 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 Haleniaelliptica require multiple chromatographic steps on silica gel, C18, and Sephadex LH-20, etc [3,4].

In this study, we successfully employed HSCCC to isolate and purify four xanthone glycosides from Haleniaelliptica at high purity rates. The experiment obtained the compounds from a 100 mg of crude sample in aone-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 wasan 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 systemsSolvent system (V/V/V/V) K（Partition coefficient）

C1 C2 C3 C4ethyl 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 1.93ethyl acetate- n-butanol-water (4:4:5) 0.53 0.79 2.34 1.56chloroform-methanol-water (4:5:4) 0.93 0.47 0.39 0.34chloroform-methanol-water (5:4:2) 1.03 0.62 0.56 0.48chloroform-methanol-water (5:3:2) 1.80 0.95 0.64 0.41ReferencesReferencesReferencesReferences1. 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.



P-P-P-P-33334444AAAA CombinationCombinationCombinationCombination ofofofof UltraUltraUltraUltrahighhighhighhigh pressurepressurepressurepressure----AAAAssistedssistedssistedssisted EEEExtractionxtractionxtractionxtraction withwithwithwith HHHHigh-igh-igh-igh-SSSSpeedpeedpeedpeed

CCCCounter-ounter-ounter-ounter-CCCCurrenturrenturrenturrent CCCChromatographyhromatographyhromatographyhromatography forforforfor thethethethe PreparationPreparationPreparationPreparation ofofofof CantharidinCantharidinCantharidinCantharidin fromfromfromfromthethethethe blisterblisterblisterblister beetlebeetlebeetlebeetle

XiaojingXiaojingXiaojingXiaojing LinLinLinLin 1111,,,, HongjingHongjingHongjingHongjing DongDongDongDong 1,21,21,21,2,,,, Yan-lingYan-lingYan-lingYan-ling GengGengGengGeng 1111,,,, FengFengFengFeng LiuLiuLiuLiu 1111,,,, DaijieDaijieDaijieDaijie WangWangWangWang 1111,,,, XiaoXiaoXiaoXiao WangWangWangWang 1*1*1*1*

1 Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong250014, China

2 College of Pharmacy, Shandong University of Traditional Chinese Medicine, University Science andTechnology Park in Changqing, Jinan, Shandong 250355, China

*Correspondence: Dr. Xiao Wang, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19Keyuan Street, 250014 Jinan, P. R. China. E-mail: [email protected], Fax: +86-531-82964889

AbstractAbstractAbstractAbstract 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.

KeywordKeywordKeywordKeyword: Cantharidin; High-speed counter-current chromatography; Ultrahigh

pressure-assisted extraction


PPPP----33335555CCCContinuousontinuousontinuousontinuous separationseparationseparationseparation ofofofof capsaicincapsaicincapsaicincapsaicin andandandand dihydrocapsaicindihydrocapsaicindihydrocapsaicindihydrocapsaicin usingusingusingusing sequentialsequentialsequentialsequential

centrifugalcentrifugalcentrifugalcentrifugal partitionpartitionpartitionpartition chromatographychromatographychromatographychromatographyJohannesJohannesJohannesJohannes Goll,Goll,Goll,Goll, AndreasAndreasAndreasAndreas Frey,Frey,Frey,Frey, MirjanaMirjanaMirjanaMirjana Minceva*Minceva*Minceva*Minceva*

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

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

Keywords:Keywords:Keywords:Keywords: sequential centrifugal partition chromatography, method development, process design

In sequential centrifugal partition chromatography (sCPC) a liquid-liquid biphasic system is used to performcontinuous cyclic separations of a feed mixture in two fractions.(1) One sCPC cycle includes two steps; oneperformed in ascending mode and the other in descending mode. The feed is introduced continuously betweentwo adjacent columns of the sCPC, while the products are collected sequentially at the opposite ends of thecolumns. During the ascending step the upper phase is used as a mobile phase and carrier (solvent) of the feedcomponents, 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 ofseparation. This makes this technique very suitable for preparative application, especially in the cases ofexpensive fine chemicals, which are difficult to separate because of their physico-chemical similarity. Oneexample is the separation of capsaicin from dihydrocapsaicin, two bioactive components, which are widelyused 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 wasdone by a priori prediction of the partition coefficients of both components in the systems of the Arizonasolvent system family using COSMO-RS (“Conductor-like Screening Model for Real Solvents).(3) Thisapproach reveals that Arizona system N, containing equal volume fractions of water, heptane, ethyl acetate andmethanol, is the most suitable system. Subsequently, the partition coefficients of capsaicin anddihydrocapsaicin in the selected biphasic system were determined from a pulse injection experiments. Thepulse injection elution profiles obtained in ascending and descending mode using different mobile phase flowrates 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 twocolumns of 125 ml was used. The sCPC runs were carried out at room temperature, atmospheric pressure and arotational speed of 1700 rpm, using equal volumes of the upper and lower phase in the sCPC columns. Theconcentration of capsaicin and dihydrocapsaicin in the feed was 5 g/l, each. The duration of the two steps of thesCPC cycle and the feed and mobile phase flow rates, in each step of the sCPC cycle, were selected using theapproach 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 processwas modeled using the cell model. For the simulation of the sCPC operation performances the experimentallyderived 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 dihydrocapsaicinwas achieved. Furthermore, the agreement between the simulated and experimentally achieved sCPCperformance was more than satisfactory.ReferencesReferencesReferencesReferences

1. Hopmann E.; Minceva M. J.Chromatogr., A 2012,2012,2012,2012, 1229, 140-147.2. Peng A.; Ye H.; Li X.; Chen L. J. Sep. Sci. 2009,2009,2009,2009, 32, 2967-2973.3. Hopmann E.; Frey A.; Minceva M. J.Chromatogr., A 2012,2012,2012,2012, 68-76.4. Völkl J.; Arlt W.; Minceva M. AIChE J, 2012,2012,2012,2012, in press DOI 10.1002/aic.13812.



P-P-P-P-33336666SSSSeparationeparationeparationeparation ofofofof sixsixsixsix ginsenosidesginsenosidesginsenosidesginsenosides fromfromfromfrom panaxpanaxpanaxpanax ginsengginsengginsengginseng usingusingusingusing high-performancehigh-performancehigh-performancehigh-performance

counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyYiYiYiYi LiLiLiLi1,*1,*1,*1,*,,,, FedericoFedericoFedericoFederico BuonocoreBuonocoreBuonocoreBuonocore2222,,,, JamesJamesJamesJames BarkerBarkerBarkerBarker 2222,,,, StephenStephenStephenStephen JJJJ BartonBartonBartonBarton2222,,,, IanIanIanIan A.A.A.A. SutherlandSutherlandSutherlandSutherland1111

andandandand SvetlanaSvetlanaSvetlanaSvetlana IgnatovaIgnatovaIgnatovaIgnatova1111

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:Keywords:Keywords: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 diversebiological activities. In the present work, a fast and convenient method for the separation and purification of sixginsenosides from Panax ginseng by high-performance counter-current chromatography was successfullydeveloped. One gradient method in normal phase mode was applied in the first separation step for the isolationof ginsenosides with an EtOAc–BuOH–aqueous 5mM ammonium acetate solvent system. The compositionratio of mobile phase changed from 3.5:0.5:4 to 2.5:1.5:4. Ginsenosides Rd, Rg1, Rb2 and Rb1 (columncontent) were separated in less than 100 minutes with purities of 92%, 90%, 80% and 90% respectively andwith Re/Rc co-eluting. The final retention of stationary phase was 77.6%. Methylenechloride–methanol–isopropanol–aqueous 5mM ammonium acetate (6:2:3:4, v/v) solvent system was used for theisocratic separation of ginsenosides Re and Rc in the second separation step. The purity of ginsenosides Re andRc was assessed by HPLC–DAD to be over 90%. These ginsenosides structures were characterized byelectrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. Ammoniumacetate was used to shorten the separation time and eliminate emulsification all together. The salt can beremoved by re-dissolving the sample using acetone.

Figure 1.The fractogram of ginsenosides separation using EtOAc–BuOH–aqueous 5mM ammonium acetate solvent system onDE-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 5mMammonium acetate (6:2:3:4) on DE-Spectrum. Coil volume: 73 mL


P-P-P-P-33337777IsolationIsolationIsolationIsolation andandandand PurificationPurificationPurificationPurification ofofofof PheromonesPheromonesPheromonesPheromones fromfromfromfrom GroundGroundGroundGround BeetlesBeetlesBeetlesBeetles bybybyby

Counter-CurrentCounter-CurrentCounter-CurrentCounter-Current ChromatographyChromatographyChromatographyChromatographyWeiWeiWeiWei Jiang,Jiang,Jiang,Jiang, BinBinBinBin Wu*Wu*Wu*Wu*

Institute of Marine Biology and Natural Products, Department of Ocean Science andEngineering, 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–ethylacetate–methanol–water was applied to the isolation and purification of pheromones from ground beetles. Onenew and one known sex pheromones were obtained from the crude sample in a one-step separation. Theirpurities were determined by HPLC. The structures of these two compounds were identified by 1D and 2DNMR and MS means. Furthermore, the absolute configurations were determined by analysis of their CDspectrum.

.

Figure 1.Sex pheromones from ground beetles


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

DELETE THIS AND THE FOLLOWING TEXT BEFORE SUBMISSION

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 andnot in compliance with these template guidelines or missing sections will be returned. Use RTF file format only andsubmit by e-mail to [email protected]. Note that abstracts sent by fax or mail cannot be considered.

OH

HO

O

O

OH

HO

O

O

1111 2222


P-P-P-P-33338888

SeparationSeparationSeparationSeparation ofofofof bioactivebioactivebioactivebioactive compoundscompoundscompoundscompounds fromfromfromfrom DarkDarkDarkDark teateateatea usingusingusingusing HSCCCHSCCCHSCCCHSCCC target-guidedtarget-guidedtarget-guidedtarget-guidedbybybyby αααα-glucosidase-glucosidase-glucosidase-glucosidase inhibitoryinhibitoryinhibitoryinhibitory activityactivityactivityactivity

QiongxianQiongxianQiongxianQiongxian YeYeYeYe1111,,,, ShengShengShengSheng YinYinYinYin1111,,,, ZhiminZhiminZhiminZhimin ZhaoZhaoZhaoZhao1111,,,, DepoDepoDepoDepo YangYangYangYang1111,,,, LongpingLongpingLongpingLongping ZhuZhuZhuZhu1111,,,, LeslieLeslieLeslieLeslie BrownBrownBrownBrown2222,,,, DongmeiDongmeiDongmeiDongmeiWangWangWangWang1111****

1 School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China2 AECS-QuikPrep Ltd, Bridgend, S. Wales, CF31 4XZ, UK

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

Keywords:Keywords:Keywords:Keywords: dark tea; HSCCC; polyphenol; α-glucosidase inhibitor

Recent studies showed that post-fermented tea-dark tea possesses beneficial hyperglycemia activity [1,2]. Inthis study, 7 fractions were obtained from the 60% ethanolic extract of dark tea by D101 macroporousabsorbent column chromatography, and their inhibitory activities against α-glucosidase were assayed. As theresults, Fr. 6 showed strongest activity and was chosen to further separation by HSCCC. Solvent systems wereexamined according to partition coefficients [3] of target compounds (Figure 1, Table 1), and ethylacetate-n-butanol-methanol-water-acetic acid (4:1:1:4:0.1, v/v/v/v) system was selected. The experimentalparameters of HSCCC were shown in Figure 2. Under these conditions, 120 mg Fr.6 sample was introducedinto 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, 1HNMR and 13C NMR.

Table 1. The KD of the target compounds in different ratios of volume of solvent system: ethylacetate-n-butanol-methanol-water-acetic acid (EA-n-bu-M-W-AcA)

Solvent systems

(EA-n-bu-M-W-Ac

A)

KKKKDDDDaaaa

CompoundCompoundCompoundCompound 1111 CompoundCompoundCompoundCompound 2222

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


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 adultswith 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

Figure 1. HPLC chromatograms.(a) Fraction 6 from the 60% ethanolicextract of dark tea ; (b) HSCCC fraction of peak B in Figure 2; (c)HSCCC fraction of peak A in Figure 2. Conditions: column, UltimateAQ-C18, 250 mm*4.6 mm ID, 5 μm; mobile phase, acetonitrile and0.5% acetic acid aqueous at the gradient program (acetonitrile: 0-20 min,5-25%; 20-30 min, 25-30%; 30-35 min, 30-90%; 35-45 min, 90%);flow rate, 1.0 mL/min; detection wavelength, 280 nm.

Figure 2. HSCCC separation of the fraction 6 from the 60%ethanolic extract of dark tea using two phase solvent systemcomposed of ethyl acetate-n-butanol-methanol-water-acetic acid at4:1:1:4:0.1 v/v; stationary phase: upper organic phase; coil volume,120 mL; rotary speed, 850 rpm; flow rate, 1.5 mL/min; detection,280 nm; sample size,120 mg of the sample dissolved in 5 mL upperphase and 5 mL lower phase; retention of the stationary phase,58.6%.

Tel:(020)39943042



P-P-P-P-39393939AAAApplicationpplicationpplicationpplication ofofofof highhighhighhigh speedspeedspeedspeed countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography

inininin organicorganicorganicorganic synthesissynthesissynthesissynthesis purificationpurificationpurificationpurificationR.R.R.R. GGGGöööök,k,k,k, G.G.G.G. Jerz,Jerz,Jerz,Jerz, P.P.P.P. WinterhalterWinterhalterWinterhalterWinterhalter ****

* Technische Universität Braunschweig, Institute of Food Chemistry,Schleinitzstrasse 20, 38106 Braunschweig, Germany

(Fax: +49-531-391-7200, email: [email protected])Keywords:Keywords:Keywords:Keywords: high-speed countercurrent chromatography, HSCCC, organic synthesis, purification

1,1,6-Trimethyl-1,2-dihydronaphthalene (5,5,5,5, TDN), an off-flavor is found in a variety of wines, especially in Rieslingwines. 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 (4444, ionene), an educt for the synthesis of TDN (3) and3,4-didehydro-b -ionone 2222, an educt for the synthesis of known TDN precursors (2) are important compounds for theRiesling wine research. The reaction commonly used to synthesize 3,4-didehydro-b -ionone 2222 is a bromination withsubsequent debromination step, by which a double bond is inserted in the molecule. (2, 4) Ionene 4444 is synthesizedstarting from b -ionone 1111 by an iodine-catalyzed cyclization reaction (3) (Figure 1).

O 1 . N B S O

CC l4, ∆

2. N ,N -D ie thy lanil in e

p yr id in e, ∆

b y-pr odu cts

O

O

I2 , ∆

CC l4b y-pr odu cts

1111 2222 1111

1111 4444 5555

AAAA

BBBB

Figure 1. Synthesis of 3,4-didehydro-b -ionone 2222 and 1,1,6-Trimethyl-1,2,3,4-tetrahydronaphthalene 4444

Interestingly, the application of high-speed countercurrent chromatography (HSCCC) for the purification of only afew organic reaction types including catecholamines (5) and heterocycles (6) has been published. The use of thistechnique for the separation of products from organic synthesis is rare compared to the reported phytochemicalapplication of HSCCC.This work describes the application of HSCCC as an useful, fast and economic alternative for the isolation andpurification of 3,4-didehydro-b -ionone 2222 from starting material b -ionone 1111 (reaction A), and ionene 4444 from TDN 5555(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 isunsatisfactory and demanded large solvent amounts, as well as long separation times causing serious degradation ofthe 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 theoff-line injection, fractions were diluted by the factor 5.

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


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.


P-P-P-P-44440000PPPPreparativereparativereparativereparative separationseparationseparationseparation ofofofof biflavonoidsbiflavonoidsbiflavonoidsbiflavonoids fromfromfromfromGGGGarciniaarciniaarciniaarcinia kolakolakolakola heckel.heckel.heckel.heckel. seedsseedsseedsseeds bybybyby

spiralspiralspiralspiral high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography

ChrisChrisChrisChris O.O.O.O. OkunjiOkunjiOkunjiOkunjia,ca,ca,ca,c,,,, PeterPeterPeterPeter I.I.I.I. AwachieAwachieAwachieAwachieaaaa,,,, MauriceMauriceMauriceMaurice M.M.M.M. IwuIwuIwuIwuaaaa;;;;

MichelMichelMichelMichel TchimeneTchimeneTchimeneTchimeneaaaa,,,, andandandand YoichiroYoichiroYoichiroYoichiro Ito*Ito*Ito*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, [email protected]

Keywords:Keywords:Keywords: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 havebeen used in many herbal preparations. Recent pharmacological research indicates that G.kola hassignificant potential including antimicrobial, hepato-protective, bronchodilator effect, anti-inflammatoryand antidiabetic activities. G.kola, characteristically, contains high concentrations of biflavonoids,prenylated benzophenones, xanthone and kolaviron. A number of herbal products derived from G. kolaseeds has been manufactured and marketed as dietary supplements or phytomedicines1. Thus, there is needfor large quantities of the bioactive constituents of G.kola for further in vivo studies, pharmacologicaltesting, chemical optimization or quality control. The key steps in this preparative separation of thebiflavonoids are the optimization of solvent combinations and addition of low concentration oftrifluoroacetic acid (TFA) to the solvent system. Using an optimized two-phase solvent system composedof 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 (1111) GB-1a (2222), GB-1 (3333), GB-2 (4444), kolaflavonone (5555), weresuccessively 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 structuresof these biflavanones were elucidated by a combination of spectrometric methods and comparison withreference standards. In comparison to earlier HSCCC separation3333, the results revealed that the new spiralHSCCC system yielded high partition efficiency with the best stationary phase retention and a shortelution time.


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.



PPPP-41-41-41-41SeparationSeparationSeparationSeparation ofofofof anti-tumoranti-tumoranti-tumoranti-tumor constituentsconstituentsconstituentsconstituents fromfromfromfrom KalopanaxKalopanaxKalopanaxKalopanax septemlobusseptemlobusseptemlobusseptemlobus (Thunb.)(Thunb.)(Thunb.)(Thunb.)KoidzKoidzKoidzKoidz bybybyby silicasilicasilicasilica gelgelgelgel columncolumncolumncolumn andandandand high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography

JingJingJingJing Xu,Xu,Xu,Xu, XueliXueliXueliXueli Cao*,Cao*,Cao*,Cao*, LuLuLuLu Yin,Yin,Yin,Yin, ChaoChaoChaoChao Cheng,Cheng,Cheng,Cheng, HongHongHongHong RenRenRenRen

Beijing Key Lab of Plant Resource Research and Development, School of Food and Chemical Engineering, BeijingTechnology and Business University, Beijing 100048, China.

*E-mail: [email protected]；Fax: 00-86-10-68984898Keywords:Keywords:Keywords: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 inhibitoryactivity against A-549 human lung cancer cells in vitro and against Lewis lung cancer in vivo in our recentresearches. In order to investigate potential anti-tumor compounds from K. septemlobus, the ethanol extract ofthe stem bark of K. septemlobus was fractioned by extraction with petroleum ether, ethyl acetate and n-butanolsuccessively. According to the results of MTT assay, the petroleum ether fraction was subjected to furtherseparation 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 etherextract. Then, 12.2mg of (-)-asarinin (4) were obtained from fraction A by high-speed counter currentchromatography (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 separatedby 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 wereall over 95% determined by HPLC. The chemical structures were confirmed by 1H-NMR, 13C-NMR andESI-MS. Further MTT assay proved the anti-tumor activity of luvangetin (3) against A-549 human lung cancercells in a dose-dependent manner with IC50 values at 4.28 μg/mL. The anti-tumor activities of others are underinvestigation.


1. Ge Bai, Xueli Cao, Hong Zhang, Junfeng Xiang, Hong Ren, Li Tan, Yalin Tang. Direct screening of G-quadruplex ligandsfrom 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 RodriguesFilho, Maria Fátima G. Fernandes da Silva, Paulo Cezar Vieira, João Batista Fernandes. Isolation of xanthyletin, an inhibitorof ants’ symbiotic fungus, by high-speedcounter-current chromatography. J. Chromatogr. A.1216 (2009) 4307–4312.

This project is financially supported by Beijing Natural Science Foundation.

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



P-P-P-P-44442222EffectiveEffectiveEffectiveEffective andandandand PreparativePreparativePreparativePreparative SeparationSeparationSeparationSeparation ofofofof BioactiveBioactiveBioactiveBioactive FlavonoidsFlavonoidsFlavonoidsFlavonoids fromfromfromfrom

GynostemmaGynostemmaGynostemmaGynostemma pentaphyllumpentaphyllumpentaphyllumpentaphyllum TeaTeaTeaTea UsingUsingUsingUsing Elution-ExtrusionElution-ExtrusionElution-ExtrusionElution-Extrusion Counter-CurrentCounter-CurrentCounter-CurrentCounter-CurrentChromatographyChromatographyChromatographyChromatography

ZheZheZheZhe ChenChenChenChen 1111,,,, YanbinYanbinYanbinYanbin LuLuLuLu 2,2,2,2, ****1 Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University,

Hangzhou 310006, China2 College of Food and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China

*fax +86-571-88071024-7587, e-mail: [email protected]

Keywords:Keywords:Keywords:Keywords: Counter-current chromatography; Elution-extrusion; Preparative chromatography; Gynostemmapentaphyllum; 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 partis 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 andpharmacological studies on this species, because of the unique structures and various biological activities of therelative compounds.

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

Fig.Fig.Fig.Fig. 1111 (A) EECCC separation of G. pentaphyllum extracts using a 180 mL CCC column. Biphasic liquid system: n-hexane/ethylacetate/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.


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.


P-4P-4P-4P-43333PreparativePreparativePreparativePreparative separationseparationseparationseparation ofofofof polyphenolspolyphenolspolyphenolspolyphenols bybybyby high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current

chromatographychromatographychromatographychromatography

ZhenhuaZhenhuaZhenhuaZhenhua Li,Li,Li,Li, MinhuiMinhuiMinhuiMinhui Li*Li*Li*Li*

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

Abstract:Abstract:Abstract:Abstract:

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

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

O

OH

OH

OH

OH

O

HO HO

OH

O

OH

OH

OH

OH

HOO

OH

HO

22221111

Figure 1.Polyphenols from Vitis quinguangularis


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. Nunogaki3. Y. Ito, J. Sandlin, W.G. Bowers J. Chromatogr., 244 (1982), p. 247

DELETE THIS AND THE FOLLOWING TEXT BEFORE SUBMISSION

The Scientific Committee of CCC 2012 will review all abstracts. Abstracts must be submitted before the June1, 2012 deadline. Abstract contents are the responsibility of the authors. Abstracts exceeding the one-page limitand not in compliance with these template guidelines or missing sections will be returned. Use RTF file formatonly and submit by e-mail to [email protected]. Note that abstracts sent by fax or mail cannot beconsidered.


P-P-P-P-44444444IIIIsolationsolationsolationsolation ofofofof thethethethe cycloartanecycloartanecycloartanecycloartane glycosidesglycosidesglycosidesglycosides fromfromfromfrom SSSSutherlandiautherlandiautherlandiautherlandia frutescensfrutescensfrutescensfrutescens bybybyby

HSCCCHSCCCHSCCCHSCCC usingusingusingusing thethethethe spiralspiralspiralspiral tubingtubingtubingtubing supportsupportsupportsupport columncolumncolumncolumnKoreyKoreyKoreyKorey BrownsteinBrownsteinBrownsteinBrownstein1111,,,, GeorgeGeorgeGeorgeGeorge E.E.E.E. RottinghausRottinghausRottinghausRottinghaus1111,,,, MarthaMarthaMarthaMartha KnightKnightKnightKnight2222,,,, YoichiroYoichiroYoichiroYoichiro ItoItoItoIto3333,,,, WilliamWilliamWilliamWilliam

R.R.R.R. FolkFolkFolkFolk1*1*1*1*

1. University of Missouri, Columbia MO USA 2. CC Biotech, Rockville MD USA3. National Institutes of Health, Bethesda MD USA

*corresponding author: [email protected],Keywords:Keywords:Keywords: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 intraditional medicines for modu-lating immunity, stress and chronic diseases, cancers and symptoms of HIVandother 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 bychromatography on silica gel and reverse-phase silica gel (1, 2), but the procedure is laborious andcapacity-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 withgood yield. A planetary centrifuge (Conway Centrichrom) mounted with an STS rotor (CC Biotech) (5) filled with 1.6 mm ID FEPtubing, 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 leafpowder was injected and the lower phase pumped at 1mL/min, with 820 rpm and 4-min fractions collected. Elution mode wasL-i-H and stationary phase retention was 55%. After freeze-drying and resuspension in MeOH, fractions were analyzed on TLCplates 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 asolvent system without n-butanol (EMW-8:1:7) caused the sutherlandiosides to elute toward the solvent front, and increasing thecontent of n-butanol (EBMW-19:5:4:22) caused the sutherlandiosides to elute very late. Fractions were quantitatively and qualitatively analyzedby 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 inbulk material (1). The other sutherlandiosides appeared to be recovered in similar yields. The efficiency andscalability of HSCCC for purification of the sutherlandiosides allows for examination of the biological andbiochemical activities of these novel secondary metabolites.Supported by NIH/NCCAM/ODS grants 5U19AT003264 and 1P50AT600273.ReferencesReferencesReferencesReferences1. 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.



P-P-P-P-44445555

PreparativePreparativePreparativePreparative separationseparationseparationseparation ofofofof alkaloidalkaloidalkaloidalkaloidssss fromfromfromfrom AconitumAconitumAconitumAconitum carmichaelicarmichaelicarmichaelicarmichaeli bybybybypH-zone-refiningpH-zone-refiningpH-zone-refiningpH-zone-refining counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography

LiuLiuLiuLiu DahuiDahuiDahuiDahui1111,,,, ShuShuShuShu XikaiXikaiXikaiXikai2222,,,, LiuLiuLiuLiu WeiWeiWeiWei2222,,,, WangWangWangWang XiaoXiaoXiaoXiao2,3*2,3*2,3*2,3*,,,, YangYangYangYang BinBinBinBin3333,,,, HuangHuangHuangHuang LuqiLuqiLuqiLuqi33331. 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:Keywords:Keywords: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 Aconitumcarmichaeli Debx; Condition: Two-phase solvent system: petroleum ether-ethyl acetate- methanol-water (5:5:1:9, v/v); Stationaryphase: the upper organic phase; mobile phase: the lower phase; flow rate: 2.0 ml/min; revolution speed: 850 rpm; detectionwavelength: 254 nm; sample size: 2.3 g of sample dissolved in 5 ml of the lower phase without hydrochloric acid and 15 ml of theupper phase of 5:5:1:9 system; retention of the stationary phase: 35 %.

ReferencesReferencesReferencesReferences1. F. Gao; Shou-An Zhu; Shi-Jun Xiong; Acta Cryst; 2010201020102010; E66, P.1342.

2. Van Cao; Xiaofei Chen; Di·Va Liil; Xin Dong; Guo-Qing Zhang; Vi Feng Chai; J PharmAnal; 2011201120112011; 1(2), P.125-134.

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



app:ds: alkaloid


app:ds: alkaloid

app:ds: triethylamine

app:ds:hydrochloric acid

app:ds: alkaloid

app:ds:compound

app:ds:hydrochloric acid


P-P-P-P-44446666PPPPreparativereparativereparativereparative separationseparationseparationseparation andandandand purificationpurificationpurificationpurification ofofofof hypocrellinshypocrellinshypocrellinshypocrellins fromfromfromfrom shiraiashiraiashiraiashiraia

bbbbambambambambusicolausicolausicolausicola bybybyby high-sphigh-sphigh-sphigh-speedeedeedeed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatography andandandand preparativepreparativepreparativepreparativehighhighhighhigh performanceperformanceperformanceperformance liquidliquidliquidliquid chromatographychromatographychromatographychromatography

MeixiaMeixiaMeixiaMeixia Xu,Xu,Xu,Xu, FengFengFengFeng Liu,Liu,Liu,Liu, XiuliXiuliXiuliXiuli Ma,Ma,Ma,Ma, HengqiangHengqiangHengqiangHengqiang Zhao,Zhao,Zhao,Zhao, JianhuaJianhuaJianhuaJianhua Liu,Liu,Liu,Liu, XiaoXiaoXiaoXiao Wang*Wang*Wang*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:Keywords:Keywords:Keywords: Shiraia bambusicola; hypocrellins; high-speed counter-current chromatography; preparative highperformance liquid chromatography;

Shiraia bambusicola P. Hennigs, an ascomycete parasitic on bamboo twigs, has been commonly used as medicinalfungi for treatment of rheumatism and pneusomia in traditional Chinese medicine (1). The fungus is of great medicalimportance mainly because of its metabolite- hypocrellins which exhibit photodynamic activity towards bacteria andfungi (2). In previously study, perylenequinone pigments hypocrellin A-D and shiraiachrome A-C have been isolatedfrom S. bambusaicola (3,4,5). In consideration of the biological activities of hyprocellins, it is necessary to developan efficient method to separate and purify large quantities of hyprocellins with high purity for furtherpharmacological research.

In this research, a HSCCC-PHPLC method was successfully developed for the preparative separation andpurification of hypocrellins from Shiraia bambusicola. The crude sample was extracted by 95% industrial ethanolwith percolating extraction. It was isolate by HSCCC. Then, PHPLC was used to isolate hypocrellin A andhypocrellin C .

A

B

Figure 2Figure 1

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: 850rpm.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; detectionwavelength: 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 and12.7mg hypocrellin B were obtained with the purity of 96.85%, 95.41%, 96.58%, detected by HPLC. The structuresof 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 bythe HSCCC-PHPLC method. It showed that HSCCC-PHPLC was an efficient method for the isolation andpurification of the hypocrellins from Shiraia bambusicola. And it is of great value on the further development andutilization of Shiraia bambusicola resources.

ReferencesReferencesReferencesReferences1. Liu,B. Chinese medical fungi, 2nd ed., Shanshi People's Press: Shanshi, 1978197819781978.2. Wang HK; Xie JX; Chang JJ; Hwang KM; Liu SY; Lawrence MB; Jing JB; Lee KH. J. Med Chem. 1992199219921992, 35, 2721–2727.3. Kishi T; Tahara S; Taniguchi N; Tsuda M; Tanaka C; Takahashi S. Planta Med. 1991199119911991, 57, 376–379.4. Lizhen Fang; Chen Qing; Hongjun Shao; Yidong Yang; Zejun Dong; Fei Wang; Wei Zhao; Wanqiu Yang; Jikai Liu. J. Antibiot. 2006200620062006, 59,

351–354.5. Houming Wu; Xiafei Lao; Qiwen Wang; Renlong Lu. J. Nat. Prod. 1989198919891989, 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.



P-P-P-P-44447777Separation,Separation,Separation,Separation, identificationidentificationidentificationidentification andandandand purificationpurificationpurificationpurification ofofofof activeactiveactiveactive constituentsconstituentsconstituentsconstituents ofofofof

PPPPolygonumolygonumolygonumolygonum cuspidatumcuspidatumcuspidatumcuspidatum sieb.sieb.sieb.sieb. etetetet zucc.zucc.zucc.zucc. bybybyby HPLC-PDA-ESI-MSHPLC-PDA-ESI-MSHPLC-PDA-ESI-MSHPLC-PDA-ESI-MSnnnn andandandand HSCCCHSCCCHSCCCHSCCC

FangyuanFangyuanFangyuanFangyuan GaoGaoGaoGaoa,a,a,a, bbbb,,,, GaoGaoGaoGao FangFangFangFang a,a,a,a, b,b,b,b, cccc,,,, TingtingTingtingTingtingTingting ZhouZhouZhouZhoua,a,a,a, bbbb,,,, JiJiJiJi LiLiLiLia,a,a,a, bbbb,,,, GuorongGuorongGuorongGuorong FanFanFanFana,a,a,a, b,b,b,b, ****

a, * Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, No. 325Guohe Road , Shanghai 200433, PR China

b Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military MedicalUniversity, No. 325 Guohe Road, Shanghai 200433 P.R China

c Department of Pharmacognosy, School of Pharmacy, Anhui University of Traditional Chinese MedicineKeywordsKeywordsKeywordsKeywords: Polygonum cuspidatum; HPLC-PDA-ESI-MSn; HSCCC

Polygonum cuspidatum Sieb. et Zucc. is a well known traditional Chinese medicine commonly used fortreatment 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 theP.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 totalof fourteen bioactive components were identified by HPLC-PDA-ESI-MSn. The typical chromatogram isshown in Figure 1.

An HSCCC was applied to prepare and purify the hydrophobic compounds from the 75% ethanol extract ofP.cuspidatum by ultrasound assisted microwave extraction. The HSCCC separation was performed on ananalytical 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 -ethyacetate-methanol-water（3:5:9:3 v/v）. During separating in the head to tail mode, the instrument parameter wasadjusted to 1800 rpm for rotation, 25 ℃ for temperature, 1 mL/min for flow rate and 254 nm for detectionwavelength.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 byHSCCC based on the

identification of these fourteen constituents by HPLC-PDA-ESI-MSn. The application of these combinationtechniques mentioned above can be utilized in the field of the quality control of TCMs.ReferencesReferencesReferencesReferences1. Dong, J.; Wang, H.; Wan, L.; Hashi, Y.; Chen, S., Identification and determination of major constituents in Polygonum cuspidatum Sieb. etZucc. by high performance liquid chromatography/electrospray ionization-ion trap-time-of-flight mass spectrometry. Chinese Journal ofChromatography 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 etRadix. 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 cuspidatumSieb. 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 fromPolygonum cuspidatum. J. Chromatogr. A 2001, 907 (1-2), 343-346.

Figure 1. HPLC chromatogram of the 75% ethanol

extract of Polygonum cuspidatumFigure 2. The HSCC chromatogram of the 75% ethanol extract

of Polygonum cuspidatum.


P-P-P-P-44448888PurificationPurificationPurificationPurification ofofofof aaaa smallsmallsmallsmall lectinlectinlectinlectin fromfromfromfrom aaaa hydroponichydroponichydroponichydroponic cultureculturecultureculture mediamediamediamedia usingusingusingusing

centrifugalcentrifugalcentrifugalcentrifugal partitionpartitionpartitionpartition chromatographychromatographychromatographychromatographyLukaszLukaszLukaszLukasz Grudzien*Grudzien*Grudzien*Grudzien*aaaa,,,, DerekDerekDerekDerek FisherFisherFisherFisheraaaa,,,, LuisaLuisaLuisaLuisa dededede MoraesMoraesMoraesMoraes MadeiraMadeiraMadeiraMadeirabbbb,,,, JulianJulianJulianJulian MaMaMaMabbbb,,,, IanIanIanIan

SutherlandSutherlandSutherlandSutherlandaaaa,,,, IanIanIanIan GarrardGarrardGarrardGarrardaaaaa 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 alectin from other proteins that were co-secreted into a hydroponic medium. Rhizosecretion is often used in theproduction of recombinant proteins [1]. The process involves expression of a protein in the roots of a plant andits secretion to a hydroponic media. It has considerable advantage over the extraction of recombinant proteinsdirectly from plant tissues as this latter process involves plant harvesting, tissue maceration and subsequentprotein purification. Not only is this process expensive and time consuming, but it can cause the release ofmany plant contaminants including proteolytic enzymes. Although rhizosecretion eliminates these particulardrawbacks, it does creates its own challenges: the yield of proteins obtained from rhizosecretion can be as lowas 10μg/ml of the hydroponic media and therefore proteins produced using this method need to be concentratedfrom a large volume of a hydroponic media.As part of studies to develop a method to extract a lectin proteinfrom 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 smallmolecules. The concentrated material was then processed on CPC to purify the target protein from impuritieswhich were mainly other proteins. Due to its gentle nature, ATPS was selected as the phase system. Therefore, arange of two phase systems was examined to find one which would provide differential partitioning of thetarget 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 thepolymer and salt (12% and 18%), polymer type (polyethylene glycol (PEG) and poly(ethyleneglycol-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 thesystem (3.0 and 7.0), concentration of sodium chloride (0 and 5%) and concentration of sodium perchlorate (0and 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 twoATPSs which provided differential partitioning of the target protein and impurities. Both ATPSs were used asthe 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.

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


P-P-P-P-49494949PPPPurificationurificationurificationurification ofofofof luteinluteinluteinlutein fromfromfromfrom kalekalekalekale bybybyby highhighhighhigh speedspeedspeedspeed countercurrentcountercurrentcountercurrentcountercurrent chromatographychromatographychromatographychromatography

PingxiaoPingxiaoPingxiaoPingxiao DuanDuanDuanDuan1111,,,, QingqinQingqinQingqinQingqin XuXuXuXu2222,,,, LeiLeiLeiLei ZhangZhangZhangZhang1111,,,, XiangdongXiangdongXiangdongXiangdong Wang*Wang*Wang*Wang*1111

*1 Food Science and Engineering,College of Engineering,Shan Xi Normal University. [email protected] Food Science and Engineering, College of Engineering, Shan Xi Normal University

2 Analytical Chemistry, Analysis and Testing Centre, Shan Xi Normal University

Keywords:Keywords:Keywords: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 mayserve to protect the retina from the ionizing effect of blue light since it is accumulated in human retina. Asa 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 fromkale extract.

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

0 2 4 6 8 10 12 14 16-500

0

500

1000

1500

2000

Fig. 1 HPLC chromatogram of lutein fraction from HSCCC separation


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

http://en.wikipedia.org/wiki/Cabbage

http://en.wikipedia.org/wiki/Brassica_oleracea

http://en.wikipedia.org/wiki/Acephala_Group

http://en.wikipedia.org/wiki/Lutein

http://en.wikipedia.org/wiki/Food_additive

http://en.wikipedia.org/wiki/E_number

http://en.wikipedia.org/wiki/Lutein

http://en.wikipedia.org/wiki/Food_additive

http://www.iovs.org/cgi/content/full/42/8/1873

http://www.iovs.org/cgi/content/full/42/8/1873


P-5P-5P-5P-50000NNNNewewewewHHHHyphenatedyphenatedyphenatedyphenated CPC-HPLC-DAD-MSCPC-HPLC-DAD-MSCPC-HPLC-DAD-MSCPC-HPLC-DAD-MS strategystrategystrategystrategy forforforfor simultaneoussimultaneoussimultaneoussimultaneous isolation,isolation,isolation,isolation,

analysisanalysisanalysisanalysis andandandand identificationidentificationidentificationidentification ofofofof phytochemicals.phytochemicals.phytochemicals.phytochemicals.ThomasThomasThomasThomas MichelMichelMichelMichel1111,,,, EmilieEmilieEmilieEmilie DestandauDestandauDestandauDestandau1111,,,, LaLaLaLaëëëëtitiatitiatitiatitia FougFougFougFougèèèèrererere1111,,,, GregoireGregoireGregoireGregoire Audo*Audo*Audo*Audo*2222,,,, ClaireClaireClaireClaire

ElfakirElfakirElfakirElfakir1111

1Institute of Organic and Analytical Chemistry, Université d’Orléans–CNRS, UMR CNRS 7311, BP 67059, 45067Orléans Cedex 2, France

2Armen instrument, 16 rue Ampère, 56890 Saint Avé, France. [email protected]

Keywords:Keywords:Keywords: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.



P-5P-5P-5P-51111ImpuritiesImpuritiesImpuritiesImpurities preparationpreparationpreparationpreparation andandandand identificationidentificationidentificationidentification ofofofof sodiumsodiumsodiumsodium tanshinonetanshinonetanshinonetanshinone IIIIIIII aaaa sulfonatesulfonatesulfonatesulfonate

bybybyby HSCCCHSCCCHSCCCHSCCC andandandand LC-MSLC-MSLC-MSLC-MSnnnn

ChenChenChenChen ShanqiaoShanqiaoShanqiaoShanqiaoa,ba,ba,ba,b,,,, ZhouZhouZhouZhou TingtingTingtingTingtingTingtinga,a,a,a,*,*,*,*, LiuLiuLiuLiu ChengchuChengchuChengchuChengchu bbbb,,,, FanFanFanFan GuorongGuorongGuorongGuoronga,a,a,a,****a Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical

UniversitybCollege 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:Keywords:Keywords: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 oftraditional Chinese medicine Danshen (the root of Salvia miltiorrhiza Bunge)1. Even though this drug of 30years’ history has been investigated a lot about synthesis2, the quality control especially quantitative analysis ofthe 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 aredisproportionately low in STS bulk drug taking the advantages of high sample load, high efficiency and free ofinreversible adsorption. Moreover, another impurity was prepared by preparative HPLC after HSCCCenrichment. 6 more impurities which are too low for direct LC- MS detection were identified due to theHSCCC enrichment.The HSCCC separation was performed on a TBE- 300 A semipreparative system include an Ito coil with thecapacity of 300 mL and a temperature control module. The solvent system was composed of chloroform-nbutanol-methanol-water (3:0.2:3:2). Additionally 1%(V/V) saturated Ammonium acetate aqueous solution wasadded as an demulsifier. During separating in the head to tail mode the instrument parameter was adjusted to800 rpm for rotation, 20 ℃ for temperature, 2 mL/min for flow rate and 280 nm for detect wave length. Thefractions 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 togetherexclusively while others were recovered respectively. The 3 groups and all the singly recovered fractions wereanalysed by LC- MSn. Sodium Przewaquinone A Sulfonate (5) in purity of 96.7% and Sodium HydroxylTanshinone Sulfonate (2) with 95.3% were found in group II and III. Sodium 1, 2-dehydrotanshinone II ASulfonate (8) in 100% purity was obtained by preparative separating of group I. 6 impurities and STS (1) weredetected 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 impuritiesfrom 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.


1. Sun, A.; Zhang, Y.; Li, A.; Meng, Z.; Liu, R. Extraction and preparative purification of tanshinones from Salvia miltiorrhizaBunge 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 ofDan-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 inSodium Tanshinone IIA Sulfonate. Journal of Liquid Chromatography & Related Technologies 2009, 32, 2346-2360.

Figure 1. The HSCCC chromatography that the3 groups of fractions was pooled togetherrespectively

Figure 2. The conjectured structure of STS and the impurities



P-5P-5P-5P-52222MethodMethodMethodMethod optimisationoptimisationoptimisationoptimisation forforforfor isolatingisolatingisolatingisolating purifiedpurifiedpurifiedpurified αααα-mangostin-mangostin-mangostin-mangostin fromfromfromfrom GGGGarciniaarciniaarciniaarcinia

mangostanamangostanamangostanamangostana LLLL.... rindsrindsrindsrinds usingusingusingusing HPCCCHPCCCHPCCCHPCCC

SriSriSriSri MurhandiniMurhandiniMurhandiniMurhandini1111,2*,2*,2*,2*,,,, EmilyEmilyEmilyEmily KeaveneyKeaveneyKeaveneyKeaveney3333,,,, StephenStephenStephenStephen JJJJ BartonBartonBartonBarton3333,,,, JamesJamesJamesJames BarkerBarkerBarkerBarker3333,,,, IanIanIanIan GarrardGarrardGarrardGarrard2222,,,, DerekDerekDerekDerek FisherFisherFisherFisher2222

andandandand SvetlanaSvetlanaSvetlanaSvetlana IgnatovaIgnatovaIgnatovaIgnatova2222

1. The National Agency of Drug and Food Control of the Republic of Indonesia2. Advanced Bioprocessing Centre, Brunel Institute for Bioengineering,

Brunel University London, UK3. School of Pharmacy and Chemistry, Kingston University, UK

*Correspondence should be addressed to fax: + 441895274608 and e-mail: [email protected]

Keywords:Keywords:Keywords:Keywords: α-mangostin, production, multiple injections, HPCCC

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

0

20,000,000

40,000,000

60,000,000

80,000,000

100,000,000

120,000,000

140,000,000

160,000,000

0 20 40 60 80 100 120 140 160 180 200 220 240 260

Time (min)Time (min)Time (min)Time (min)

Peak

Are

a (uV

olt*s

ec)

Peak

Are

a (uV

olt*s

ec)

Peak

Are

a (uV

olt*s

ec)

Peak

Are

a (uV

olt*s

ec)

α-Mangostin

Target 2

Target 3

Target 4

α-Mangostin Average 98% purity and 96% α-Mangostin Average 98% purity and 96% α-Mangostin Average 98% purity and 96% α-Mangostin Average 98% purity and 96%

0

20,000,000

40,000,000

60,000,000

80,000,000

100,000,000

120,000,000

140,000,000

160,000,000

180,000,000

200,000,000

220,000,000

0 10 20 30 40 50 60 70 80 90 100 110 120 130

Time (min)Time (min)Time (min)Time (min)

Peak

Are

a (uV

olt*s

ec)

Peak

Are

a (uV

olt*s

ec)

Peak

Are

a (uV

olt*s

ec)

Peak

Are

a (uV

olt*s

ec)

α-Mangostin

Target 2

Target 3

Target 4

α-Mangostin Average 99% purity and 93% yieldα-Mangostin Average 99% purity and 93% yieldα-Mangostin Average 99% purity and 93% yieldα-Mangostin Average 99% purity and 93% yield

Figure 1. HPCCC Fractogram vs. Time; 10 injections of

9.1 mg extract in 0.86 mL LP

Figure 2. HPCCC Fractogram vs. Time; 5 injections of

22.8 mg extract in 0.86 mL LP


P-5P-5P-5P-53333PPPPreparativereparativereparativereparative isolationisolationisolationisolation andandandand purificationpurificationpurificationpurification ofofofof biflavonoidsbiflavonoidsbiflavonoidsbiflavonoids fromfromfromfrom EEEEphedraphedraphedraphedra sinicasinicasinicasinica bybybyby

high-speedhigh-speedhigh-speedhigh-speed counter-currentcounter-currentcounter-currentcounter-current chromatographychromatographychromatographychromatographyJialianJialianJialianJialian Li,Li,Li,Li, LeiLeiLeiLei Fang,Fang,Fang,Fang, DaijieDaijieDaijieDaijie Wang,Wang,Wang,Wang, XikaiXikaiXikaiXikai Shu,Shu,Shu,Shu, YangLingYangLingYangLingYangLing Geng,Geng,Geng,Geng, XiaoXiaoXiaoXiao Wang*Wang*Wang*Wang*

Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong250014, 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:Keywords:Keywords: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 herbthat has been used for thousands of years (1). Ephedra is generally not used alone, but rather as part of herbalformulas, and is known to be used for treatment to autoimmune and inflammatory diseases with safe andefficient function (2). The original isolated and major active medicinal ingredients of Ephedra stems arealkaloids. Stems contain 1~3% of the total alkaloids found in Ephedra plants, with ephedrine accounting for30–90% of this total depending on the species. In addition, other active constituents, such as biflavonoids arealso found in the stem, which also showed potent bioactivies (3-5). In order to find more bioactive biflavonoidsfor further pharmacological research, it is necessary to develop an efficient method to separate and purify largequantities of single biflavonoids with high purity.

In this research, high-speed counter-current chromatography was successfully applied to the preparativeseparation and purification of biflavonoids from the stems of E. sinica. The experiment was performed with atwo-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.5:3.5:1:3, v/v). 250mg of the crude extract was purified in one-step separation, yielding 18 mg of mahuannin D, 21 mg ofmahuannin 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 theoxindole 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 bythe high-speed counter-current chromatography method, which demonstrated that HSCCC is a fast, effectiveand 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 2003200320032003, 17, 703–712.2. Ray, S.; Phadke, S.; Patel, C., Hackman, R. M.; Stohs, S. Arch. Toxicol. 2005200520052005, 79, 330–340.3. Andraws, R.; Chawla, P.; Brown, D. L. Prog Cardiovascular Dis 2005200520052005, 47, 217–225.4. Friedrich, H.; Wiedemeyer, H. Planta Medica 1976197619761976, 30, 223–231.5. Cottiglia, F.; Bonsignore, L.; Casu, L.; Deidda, D.; Pompei, R.; Casu, M.; Floris, C. Nat. Prod. Res. 2005200520052005, 19, 117–123.



P-5P-5P-5P-54444

IsolationIsolationIsolationIsolation ofofofof ChemicalChemicalChemicalChemical ConstituentsConstituentsConstituentsConstituents fromfromfromfrom IlexIlexIlexIlex rotundarotundarotundarotunda bybybyby High-SpeedHigh-SpeedHigh-SpeedHigh-SpeedCounter-CurrentCounter-CurrentCounter-CurrentCounter-Current ChromatographyChromatographyChromatographyChromatography

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, ChinabBioseparation 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] 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).

international conference on countercurrent chromatography, … 2012.pdf · 2012-08-22 · 7 th...

Documents