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Different solvent delivery methods in Counterurrent Chromatography

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Different types of solvent delivery methods from "on demand" solvent mixing to ion exchange with a particular focus on pH-zone refining

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Different solvent delivery methods in Counterurrent Chromatography

  1. 1. Solvent Delivery Methods A. On Demand Solvent Mixing B. Gradients C. pH-Zone Refining D. Ion Exchange
  2. 2. A. On Demand Solvent Mixing Anal Bioanal Chem (2005) 383: 327–340 DOI 10.1007/s00216-005-0016-7 A. Berthod . M. Hassoun . M. J. Ruiz-Angel Alkane effect in the Arizona liquid systems used in countercurrent chromatography
  3. 3. B. Gradients 1. Step gradient 1a. Polarity-Adjusted SSs 1b. Differently Formulated SSs 2. Linear gradient 3. Three phase gradient
  4. 4. Figure 3. HSCCC chromatogram of crude extract of ChanSu. HEMWat (4:6:2:4, 4:6:2.5:4 and 4:6:3.2:4 v/v) in stepwise elution; stationary phase: upper organic phase of 4:6:2:4 by volume; mobile phase: lower aqueous phase of 4:6:2:4 in 0–200min, lower aqueous phase of 4:6:2.5:4 by volume in 200– 400 min, and lower aqueous phase of 4:6:3.2:4 by volume in 400–700min, flow rate 1.5mL/min; revolution speed, 800 rpm; detection wavelength, 296 nm; separation temperature, 201C; sample size, 80 mg crude sample dissolved in 5mL of the upper phase and 5mL of the lower phase. Retention of the stationary phase: 56%. Li2010_JSS_33_1325_bufadienolides B.1a. Step Gradient: Polarity Adjusted
  5. 5. Mar. Drugs 2015, 13, 354-365; doi:10.3390/md13010354 Preparative Separation of Sulfur-Containing Diketopiperazines from Marine Fungus Cladosporium sp. Using High-Speed Counter-Current Chromatography in Stepwise Elution Mode Binbin Gu, Yanying Zhang, Lijian Ding, Shan He, Bin Wu, Junde Dong, Peng Zhu, Juanjuan Chen, Jinrong Zhang and Xiaojun Yan Figure 3. marine fungus Cladosporium sp. using stepwise elution with solvent systems A and B. Solvent system A: HEMWat (1:1:1:1), solvent system B: HEMWat (2:1:2:1); stationary phase: upper organic phase of solvent system A; mobile phase: 460 mL lower aqueous phase of solvent system A and 240 mL lower aqueous phase of solvent system B; column capacity, 300 mL, 900 rpm, 25 °C;, 2.0 mL/min; detection, 254 nm; sample size, 300 mg Sf, 68%. Peaks: 1 = cladosporin A (1) + haematocin (2); 2 = cladosporin B (3). B.1a. Step Gradient: Polarity Adjusted
  6. 6. Sf = 0.75 Aqueous stationary 5 mL/min 1200 rpm J Sep Sci. 2013 Apr;36(8):1362-1369. doi: 10.1002/jssc.201201033. Gradient elution method in centrifugal partition chromatography for the separation of a complex sophorolipid mixture obtained from Candida bombicola yeasts. Kotland A, Hadef I, Renault JH, Hamzaoui M, Martinez A, Borie N, Guilleret A, Reynaud R, Hubert J. B.1a. Step Gradient: Polarity Adjusted
  7. 7. B.1b. Step Gradient: Differently Formulated The stationary phase was water saturated with n- butanol and ethyl acetate. The stepwise elution was performed with the following solvents: n-hexane–ethyl acetate (1:1) for 80 min, n-hexane– ethyl acetate (1:2) for 80 min, n-hexane–ethyl acetate (1:4) for 80 min, ethyl acetate for 80 min, n-butanol–ethyl acetate (1:4) for 80 min, n-butanol–ethyl acetate (1:2) for 80 min, n-butanol– ethyl acetate (2:2) for 80 min, and n-butanol–ethyl acetate (2:1) for 80 min. The effluent was collected in 20-ml fractions by a fraction collector. All fractions were assayed for antioxidant activity. The fractions were combined into seven larger fractions (components) on the basis of their antioxidant activity Fig. 1. Antioxidant activity-chromatogram of fractions obtained from HSCCC separation of 600 mg ethanolic M. sempervirens leaf extract. Du2012_FC_131_1181_Mucuna
  8. 8. B.2. Linear Gradient Figure 2. The representative CCC profiles for separation of podophyllotoxin from cytotoxic fraction II with (A) and (B) isocratic elution, and (C) linear and (D) step-gradient elution by using the system of HEMWat. 1, 4-demethylpodophyllotoxin, 2, -peltatin; 3, podophyllotoxin, 4, β-peltatin. CCC separation conditions. (A) Isocratic elution with 4:6:3:7, upper phase as stationary phase and lower phase as mobile phase, retention of the stationary phase, 61.11%. (B) Isocratic elution with 4:6:4:6, upper phase as stationary phase and lower phase as mobile phase, retention of the stationary phase, 59.26%. (C) Lineargradient elution, upper phase of 4:6:3:7 as stationary phase, mobile phase: 0–120 min, 100% of the lower phase of 4:6:3:7; 120–255 min, the lower phase of 4:6:3:7 from 100% to 0 and the lower phase of 4:6:4:6 from 0 to 100%; after 255 min, 100% of the lower phase of 4:6:4:6. (D) Step- gradient elution, upper phase of 4:6:3:7 as stationary phase, mobile phase: 0– 120 min, the lower phase of 4:6:3:7; after 120 min, the lower phase of 4:6:4:6. Flow rate, 2 mL/min. Rotation speed, 900 rpm. Detection, 254 nm. Sample solution, 77 mg of the fraction II was dissolved in a solution composed of the upper and lower phases (1:1, v/v, 4 mL of the total volume). Yields and purity, (C) 1, 2.7 mg with 86.35%, 2, 8.8 mg with 99.94%, 3, 21.1 mg with 96.9%, 4, 27 mg with 99.02%; (D) 1, 2 mg with 89.19%, 2, 8.1 mg with 99.92%, 3, 20.2 mg with 96.12%, 4, 29.1mg with 98.78%. Yang2013_JSS_36_1022_podophyllotoxins
  9. 9. 3-phase gradient Filled with LP. 1000 rpm. The sample solution was loaded into the column by pumping UP0 from 0 to 20 mL/min in 3 min in the ascending mode. The UP0 was pumped for 50 min after the release of the dead volume to ensure the elution of all hydrophobic compounds. The moderately polar MP was then pumped for 33 min to elute compounds with a medium hydrophobicity. Finally, in order to recover the most hydrophilic compounds retained at the head of the column, the role of the two liquid phases was switched by pumping the aqueous LP as the mobile phase in the descending mode at 20 mL/min. Phytochem Anal. 2013 Jul-Aug;24(4):367-73. doi: 10.1002/pca.2418. Stepwise elution of a three-phase solvent system in centrifugal partition extraction: a new strategy for the fractionation and phytochemical screening of a crude bark extract. Hamzaoui M, Renault JH, Nuzillard JM, Reynaud R, Hubert J.
  10. 10. C. pH zone refining
  11. 11. pH zone refining Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito “The greatest advantage of the method is its large sample loading capacity, which exceeds 10-fold that of the standard HSCCC in the same separation column. In addition, the method provides various special features such as yielding highly concentrated fractions, concentrating minor impurities for detection, and allowing the separation to be monitored by the pH of the effluent when there are no chromophores. Selection of solvent systems and preparation of the sample are quite different from those used in the standard HSCCC technique.”
  12. 12. pH zone refining Fig. 4. Mechanism of sharp peak formation. The acid analyte is always confined around the sharp retainer border and elutes as a sharp peak with the retainer acid. R C O O H N H H H R C O O R C O OH R C O OH N H H H N H H H H N H H H F3C C O OH H N H H H R C O OH F3C C O O H N H H HH N H H H H N H H R C O OH O HO H O H O H H Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito
  13. 13. pH zone refining Fig. 5. Model experiment to demonstrate the mechanism of pH-zone refining CCC.(B) pH-zone formation of three analytes in the column (upper diagram), and the elution profile of the analytes (lower diagram). pKa (TFA) < pKa (S1) < pKa (S2) < pKa (S3) Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito
  14. 14. pH zone refining Rule 13: For an acidic analyte follow these steps: (1) A 2 ml volume of each phase and 5 microL of NH4OH (ca. 28% NH3 stock solution) (eluter) is delivered into a test tube (13mm×100 mm) or bring the pH above 10. (2) Add a small amount of the analyte (so that no significant change is made in pH), apply a stopper and vortex several times to equilibrate the contents. (3) Measure the analyte concentration in the upper and the lower phases to obtain KU/L value or Kbase. (4) If Kbase<< 1, add TFA (retainer) (ca. 20 mM) to the contents to bring the pH to around 2, and reequilibrate the contents by vortexing. (5) Using the procedure in Step (3), obtain Kacid, and if Kacid >>1, the solvent composition is suitable for separation. (6) If Kbase is not small enough, repeat the whole procedure using a less polar solvent system such as HEMWat, 1:1:1:1 in Table 1 and move upward. (7) If Kacid is not large enough, repeat the whole procedure using a more polar solvent system of terAcWat (2:2:3) in Table 2 and downward. Rule 14: For a basic analyte, substitute HCl for NH4OH at Step (1) to test Kacid<<1, and substitute triethylamine for TFA at Step (4) to test Kbase >> 1. Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito Rule 15: Start with equal molar concentrations of retainer and eluter such as 10–20mM each. Rule 16: If the settling time of the sample solution is lengthy, further dilution of the sample is recommended. Rule 17: Routinely measure the pH of the sample solution before applying it to the column. Rule 18: Leave a small amount of the stationary phase free of hydrophobic counterions at the end of the column.
  15. 15. 1. Separation of Acids C. pH zone refining
  16. 16. C.1. pH zone refining Separation of D&C Orange No. 5 by pH-zone-refining CCC. Experimental conditions: Apparatus: type-J coil planet centrifuge with a multilayer coiled column (1.6 mm ID and 320 ml capacity); solvent system: diethyl ether–acetonitrile–0.01 M aqueous ammonium acetate (pH 9 adjusted with ammonia) (4:1:5, v/v), mobile phase: lower aqueous phase; sample: 5 g of D&C Orange No. 5 dissolved in 80 ml of solvent (40 ml each phase); retainer: TFA 200 microL in the sample solution; flow rate: 3 ml/min; detection:206 nm; revolution: 800 rpm. Journal of Chromatography A, 1271 (2013) 71– 85 Journal of Chromatography A pH-zone-refining counter- current chromatography: Origin, mechanism, procedure and applications Yoichiro Ito https://cdn.shopify.com/s/files/1/0419/3361/products/265_featured_large.jpeg?v=1432937322
  17. 17. Fig. 3. PZRCCC chromatograms for the separation of the crude extract from the U. longissima Ach. Experimental conditions: (a) solvent system: PEMH (5:5:2:8, v/v); 10 mM TFA in upper organic phase and 10 mM NaOH in lower aqueous phase; (b) solvent system: PEMH (5:5:3:7, v/v); 10 mM TFA in upper organic phase and 10 mM NaOH in lower aqueous phase; (c) solvent system: PEMH (5:5:3:7, v/v); 10 mM TFA in upper organic phase and 10–20 mM NaOH in lower aqueous phase; revolution speed: 850 rpm; flow-rate: 2 mL/min; sample size: 1.2 g; UV detection wavelength: 254 nm. J Chromatogr A. 2016 Jan 4;1427:96-101. doi: 10.1016/j.chroma.2015.12.016. Optimisation and establishment of separation conditions of organic acids from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence. Sun C, Liu F, Sun J, Li J, Wang X. C.1. pH zone refining
  18. 18. CCC chromatogram for the crude extract from G. lucidum. Two-phase solvent system: PetEMWat (3:5:3:5 and 4:5:4:5), flow rate: 5.0 ml/min, revolution speed: 500 rpm, detection wavelength: 254 nm, sample size: 2 g crude extract dissolved in 15 ml upper phase (3:5:3:5). Fig. 4. Separation of mixture from peaks 3 and 4 (Fig. 3) by pH-zone-refining CCC. Above: separation of peak 3; the latter: separation of peak 4. Experimental conditions: apparatus, three multiplayer coils separation column connected in series (I.D. of the tubing = 1.6 mm, total volume = 260 ml); solvent system: chloroform–methanol–water (13:7:4), 22 mM NH4OH in upper aqueous stationary phase and 11 mM CF3COOH in lower organic phase; sample, 568 mg mixture from peak 3 were dissolved in 10 ml stationary with NH4OH added; flow rate: 2.0 ml/min; revolution speed: 800 rpm; detection wavelength: 254 nm. Food Chemistry Volume 130, Issue 4, 15 February 2012, Pages 1010–1016 Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone-refining Chun-Ru Cheng, Yi-Feng Li, Ping-Ping Xu, Rui-Hong Feng, Min Yang, Shu-Hong Guan, , De-An Guo C.1. pH zone refining
  19. 19. Separation of 500mg fatty acids from sunflower oil with pHzone- refining CCC in a normal displacement (20 mM NH3 (retainer) and 35 mM TFA (eluter)) and b reverse displacement mode (20 mM TFA (retainer) and 35 mM NH3 (eluter)) with UV/Vis signal and pH curve obtained by measuring each fraction. Experimental conditions: CCC-1000 instrument 60 mL coil volume and 2 mL/min; 1,000 rpm; solvent system: HAcMWat (40/70/14/ 5, v/v) with TFA in the upper phase and NH3 in the lower phase Anal Bioanal Chem. 2015 Jul;407(18):5503-11. doi: 10.1007/s00216-0158723-1 Overcoming the equivalent-chain-length rule with pH-zone-refining countercurrent chromatography for the preparative separation of fatty acids. Englert M, Vetter W. C.1. pH zone refining
  20. 20. J Chromatogr A. 2016 Jan 4;1427:96-101. doi: 10.1016/j.chroma.2015.12.016. Optimisation and establishment of separation conditions of organic acids from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence. Sun C, Liu F, Sun J, Li J, Wang X. C.1. pH zone refining
  21. 21. Centrifugal partition extraction in the pH-zone-refining displacement mode: an efficient strategy for the screening and isolation of biologically active phenolic compounds M Hamzaoui, JH Renault, R Reynaud, J Hubert Journal of Chromatography B 937, 7-12 The fractionation process was performed at a flow rate of 20 mL/min using a biphasic solvent system composed of terAcWat (4:1:5) in the ascending mode. Sodium hydroxide (40 mM) and trifluoroacetic acid (30 mM) were used as retainer and displacer agents, respectively. C.1. pH zone refining
  22. 22. Fig. 2. pH of the mobile phase, composition and quantities of the fraction pools from PIto PVIII recovered as a function of time during the pH-zone-refining CPE fractionation process. TTP1 sericic acid; TTP2 trachelosperogenin E; TTP3 sericoside; PP1 gallic acid; PP2 protocatechuic acid; PP3 catechin; PP4 gallocatechin; PP5 epigallocatechin; EAd23,4,3-tri-O-methylflavellagic acid; EAd3 3,3-di-O- methylellagic acid; EAd4 3,3-di-O-methylellagic acid 4-O-xylopyranoside. Centrifugal partition extraction in the pH-zone-refining displacement mode: an efficient strategy for the screening and isolation of biologically active phenolic compounds M Hamzaoui, JH Renault, R Reynaud, J Hubert Journal of Chromatography B 937, 7-12 C.1. pH zone refining
  23. 23. 2. Separation of Bases C. pH zone refining
  24. 24. Yang, F. Q., Ito, Y., J. Chromatogr. A 2001, 923, 281–285. Fig. 2. Separation of lappaconitine from a prepurified extract of A. sinomontanum Nakai. terTetWat (2:2:3, v/v), 10 mM TEA in the upper organic stationary phase and 10 mM HCl in the lower phase; sample size: 2.0 g (A), 6.5 g (B) and 10.5 g (C); flow-rate: 3 ml /min; 860 rpm; Sf: 75.8% (A), 75% (B) and 75.6% (C);. C.2. pH zone refining
  25. 25. Fig. 3. crude extract from the Macleaya cordata. : (A) ChMWat (4:3:3); 10mM HCl in upper aqueous (stationary) phase and 10mM TEA in lower phase; (B) ChEMWat (3:1:3:3); 10mM HCl in upper aqueous phase and 10mM TEA in lower organic phase; (C) ChEMWat (3:1:3:2); 10mM HCl in upper aqueous phase and 10mM TEA in lower organic phase; 850 rpm; 2 mL/min: 1.5 g; Journal of Liquid Chromatography & Related Technologies Volume 38, Issue 20, 2015 Preparative Separation of Chelerythrine and Sanguinarine from Macleaya cordata by pH-Zone-Refining Counter-current Chromatography DOI:10.1080/10826076.2015.1105257 Qian Liu, Changlei Sun, Fansheng Meng, Wei Zhao, Dapeng Li & Xiao Wang pages 1789-1793 C.2. pH zone refining
  26. 26. C.2. pH zone refining Fig. 12. Separations of a crude alkaloid extract of Crinum moorei obtained by pH-zone-refining CCC with two different elution modes. (A) Lower aqueous phase mobile and (B) upper organic phase mobile. Experimental conditions: Apparatus and column: see Fig. 10 caption; solvent system: methyl tert.-butyl ether–acetonitrile–water; stationary phase: (A) organic phase containing triethylamine at 15 mM and (B) aqueous phase containing HCl at 10 mM; mobile phase: (A) aqueous phase containing HCl at 5 mM and (B) organic phase containing triethylamine at 10 mM; flow rate: 3.3 ml/min; sample: 3 g dissolved in 30 ml of each phase; revolution: (A) 800 rpm (600 rpm until 66 ml of the mobile phase was eluted) and (B) 600 rpm throughout. Journal of Chromatography A, 1271 (2013) 71– 85 Journal of Chromatography A pH-zone-refining counter- current chromatography: Origin, mechanism, procedure and applications Yoichiro Ito
  27. 27. C.2. pH zone refining Journal of Chromatography A, 849 (1999) 421–431 Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode Jean- Hugues Renault*, Jean-Marc Nuzillard, Gae¨lle Le Croue´rour, Philippe The´penier, Monique Ze`ches-Hanrot, Louisette Le Men-Olivier Fig. 2. (A) Simulated fractogram of a mixture of vindoline, catharanthine and vincaleukoblastine plus retaining base (TEA) in the organic stationary phase. The sample was in the first theoretical plate. [TEA] 510 mM, [HCl] 58 mM, [vindoline] 5200 mM, [catharanthine] 5200 mM, [VLB] 5100 mM (concentrations in the first theoretical plate), number of theoretical plates 5150, volume of stationary phase 51.5 ml, volume of aqueous mobile phase 50.25 ml. (B) Purity and pH profile obtained for the same simulated separation.
  28. 28. Journal of Chromatography A, 849 (1999) 421–431 Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode Jean- Hugues Renault*, Jean-Marc Nuzillard, Gae¨lle Le Croue´rour, Philippe The´penier, Monique Ze`ches-Hanrot, Louisette Le Men-Olivier Fig. 3. UV chromatogram and pH profile for the separation of vindoline, catharanthine and vincaleukoblastine. Sample: ascending mode: 110 mg (0.22 mmol) of vindoline chlorhydrate, 90 mg (0.24 mmol) of catharanthine chlorhydrate and 118 mg (0.13 mmol) of vincaleukoblastine sulfate in 10 ml of aqueous stationary phase; descending mode: 100 mg (0.22 mmol) of vindoline, 84 mg (0.25 mmol) of catharanthine and 105 mg (0.13 mmol) of vincaleukoblastine in 10 ml of organic stationary phase. terAcWat (TEA, HCl) 4:1:5 (10 mM, 10mM) C.2. pH zone refining http://www.bellybytes.com/herbs/images/periwinkle.jpg
  29. 29. A B C A BC Fig. 4. pH-Zone-refining counter-current chromatogram and HPLC control for the separation of 4.0 g of alkaloid extract from Nelumbo nucifera leaves. Experiment condition: solvent system: petroleum ether–ethyl acetate–methanol–water (5:5:2:8, v/v/v/v), 10mM TEA in the upper organic stationary phase and 5mM HCl in the lower phase; retention of stationary phase: 60%; flow- rate: 1.5 mL/min; detection: 254 nm; revolution speed: 800 rpm. Fig. 3. High-speed counter-current chromatogram and HPLC control for the separation of 120mg of alkaloid extract from Nelumbo nucifera leaves. Experiment condition: solvent system: tetrachloromethane–CHCl3– methanol–0.1MHCl (1:3:3:2, v/v/v/v); retention of stationary phase: 78%; flow-rate: 1.5 mL/min; detection: 254 nm; revolution speed: 800 rpm. J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jun 1;878(19):1647-51. doi: 10.1016/j.jchromb.2010.04.020. Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography. Zheng Z1, Wang M, Wang D, Duan W, Wang X, Zheng C. C.2. pH zone refining
  30. 30. Figure 2 1.5 g of alkaloid extract from Nelumbo nucifera: terWat, 10mM TEA in the upper organic stationary phase and 5mM HCl in the lower phase; Sf – 78%; flow-rate – 1.5mL/min; 800 rpm. 1.5 g of alkaloid extract from N. nucifera HEMWat (5:5:5:5), 10mM TEA in the upper organic stationary phase and 5mM HCl in the lower phase; Sf – 70%; flow-rate – 1.5 mL/min; 800 rpm. 1.5 g of alkaloid extract from N. nucifera HEMWat (5:5:2:8), 10mM TEA in the upper organic stationary phase and 5mM HCl in the lower phase; Sf – 57%; flow- rate – 1.5 mL/ min; 800 rpm. J Sep Sci. 2010 Mar;33(4-5):539-44. doi: 10.1002/jssc.200900561. Preparative separation of alkaloids from Nelumbo nucifera Gaertn by pH-zone-refining counter-current chromatography. Wang X1, Liu J, Geng Y, Wang D, Dong H, Zhang T. C.2. pH zone refining
  31. 31. Fig. 3. Dactylicapnos scandens. Column volume : 250 mL; PetEMWat (3:7:1:9, v/v), 20 mM TEA in the upper organic stationary phase and 5 mM HCl in the lower phase; Sf 60%;: 1.5 mL/min; detection wavelength: 254 nm; 750 rpm;: 1.0 g of crude alkaloids. Fig. 4. D. scandens. column volume 500 mL; PetEMWat (3:7:1:9, v/v), 20 mM TEA in the upper organic stationary phase and 5 mM HCl in the lower phase; Sf: 60%; 1.5 mL/min; detection wavelength: 254 nm; : 750 rpm: 1.0 g of crude alkaloids. J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Dec 1;879(31):3767-70. doi: 10.1016/j.jchromb.2011.10.013. Preparative isolation of alkaloids from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column. Wang X, Dong H, Yang B, Liu D, Duan W, Huang L. C.2. pH zone refining
  32. 32. B D C Conventional HSCCC separation of fraction II (Fig. 2). Solvent system: HEMWat (7:3:6:4, v/v); revolution speed: 800 rpm; flow rate: 1.5 mL/min; sample size: 150 mg; UV detection wavelength; Sf: 80%. pH-zone-refining crude alkaloids from Stephania kwangsiensis. HEMWat (3:7:1:9, v/v); 10 mmol/L TEA in upper organic phase, 5 mmol/L HCl in lower aqueous phase: 800 rpm; 1.5 mL/min; sample size: 2.0 g; A B + C + D J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Apr 15;879(13-14):945-9. doi: 10.1016/j.jchromb.2011.02.051. Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of alkaloids from Stephania kwangsiensis. Dong H1, Zhang Y, Fang L, Duan W, Wang X, Huang L. C.2. pH zone refining
  33. 33. Large-scale separation of alkaloids from Gelsemium elegans by pH-zone-refining counter-current chromatography with a new solvent system screening method Journal of Chromatography A, Volume 1307, 13 September 2013, Pages 80-85 Lei Fang, Jie Zhou, YunLiang Lin, Xiao Wang, Qinglei Sun, Jia-Lian Li, Luqi Huang pH-zone-refining CCC separation of alkaloids from G. elegans.: HEMWat, 10 mM triethylamine in upper organic phase, 10 mM hydrochloric acid in lower aqueous phase, (A) (5:5:5:5, v/v), (B) (5:5:3:7, v/v), (C) (5:5:1:9, v/v), and (D) (3:7:1:9, v/v); 800 rpm; 2.0 mL/min; sample size: (A) 1.0 g, (B) 1.5 g, (C) 3.1 g, and (D) 4.5 g; stationary phase retention: (A) 59%, (B) 58%, (C) 50%, and (D) 47%; C.2. pH zone refining
  34. 34. Fig. 2. pH-zone-refining CCC separation of G. elegans extract. : MtBE/CH3CN/water (3:1.5:4, v/v), 20mMTEA in the upper organic stationary phase and 10mM HCl in the lower aqueous phase; sample size: 1.0 g (A) and 1.5 g (B); flow-rate: 2ml/min; detection: 254 nm; revolution speed: 850 rpm; retention of stationary phase: 58.8% (A) and 58.3% (B). J Chromatogr A. 2011 Jun 10;1218(23):3695-8. doi: 10.1016/j.chroma.2011.04.025. Preparative separation of alkaloids from Gelsemium elegans Benth. using pH-zone-refining counter-current chromatography. Su YP1, Shen J, Xu Y, Zheng M, Yu CX. C.2. pH zone refining
  35. 35. 3. Ion Exchange C. pH zone refining
  36. 36. strong ion-exchange displacement centrifugal partition chromatography (SIXCPC). Journal of Chromatography A, 1170 (2007) 44–51 Strong ion-exchange centrifugal partition chromatography as an efficient method for the large- scale purification of glucosinolates Alix Toribio, Jean-Marc Nuzillard, Jean-Hugues Renault ∗ EBuWat (Aliquat 336, NaI) 3:2:5 (160 mM, 80 mM) https://upload.wikimedia.org/wikipedia/commons/thumb/1/19/Sinalbin.svg/2000px-Sinalbin.svg.png C.3. ion-exchange displacement
  37. 37. Journal of Chromatography A, 1170 (2007) 44–51 Strong ion-exchange centrifugal partition chromatography as an efficient method for the large- scale purification of glucosinolates Alix Toribio, Jean-Marc Nuzillard, Jean-Hugues Renault ∗ The column was filled with organic stationary phase (SP) in the descending mode, at 30 mL/min and with a 300 rpm column rotation speed. The sample was then injected at 2 mL/min at 1200 rpm. NaI-free mobile phase (MP) (314 and 80mL for broccoli extract and white mustard extract, respectively) was pumped at 2 mL/min in order to allow extraction of the GSLs into the stationary phase and elution of polar impurities such as polyphenols or free sugars (40 min for S. alba extract and 157 min for B. oleracea extract). The displacing aqueous mobile phase was then pumped at 2 mL/min, and the fractions were collected every minute. C.3. ion-exchange displacement
  38. 38. C.3. ion-exchange displacement Pilot-scale ion-exchange centrifugal partition chromatography: Purification of sinalbin from white mustard seeds Journal of Separation Science. Volume 32, Issue 11, 2009 Pages 1801–1807 Alix Toribio, Jean-Marc Nuzillard, Benoît Pinel, Leslie Boudesocque, Michel Lafosse, François De La Poype, Jean-Hugues Renault http://www.apnfs.info/wp-content/uploads/2015/08/mustard-seeds-yellow-1.jpg
  39. 39. Preparative isolation of glucosinolates from various edible plants by strong ion-exchange centrifugal partition chromatography. Separation and Purification Technology, v.83, 2011 Nov 15, p.15(8) Toribio, Alix Boudesocque, Leslie Richard, Bernard Nuzillard, Jean-Marc Renault, Jean-Hugues C.3. ion- exchange displacement
  40. 40. C.3. ion-exchange displacement Preparative isolation of glucosinolates from various edible plants by strong ion-exchange centrifugal partition chromatography. Separation and Purification Technology, v.83, 2011 Nov 15, p.15(8) Toribio, Alix Boudesocque, Leslie Richard, Bernard Nuzillard, Jean-Marc Renault, Jean-Hugues

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