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Basic principles of Countercurrent Separation

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Slides presented at the annual meeting of the American Society of Pharmacognosy, Lexington Kentucky (2018). Compile basic information of the principles of countercurrent separation, choice of solvent systems, and determination of partition coefficient.
Prepared by Drs. Charlotte Simmler, Brent Friesen, Guido Pauli from the Center for Natural Product Technologies (CENAPT)

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Basic principles of Countercurrent Separation

  1. 1. What is Countercurrent Separation (CS)? It includes the following: Centrifugal Countercurrent Separation (CCS) Countercurrent Chromatography (CCC) High-speed Countercurrent Chromatography (HSCCC) Centrifugal Partition Chromatography (CPC) 1https://cenapt.pharm.uic.edu C Simmler | JB Friesen | GF Pauli CENAPT, UIC, Chicago
  2. 2. 2 A Liquid/Liquid Separation Technique….. UP LP Crude extract 2 immiscible solvents cenapt.pharm.uic.edu Simmler | Friesen | Pauli
  3. 3. 3 A Liquid/Liquid Separation Technique…using Centrifugal Forces cenapt.pharm.uic.edu Simmler | Friesen | Pauli A centrifuge is used to hold one phase stationary (SP) meanwhile the other phase (mobile) moves through, getting mixed with the SP, then separated throughout the tubing/column http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html
  4. 4. 4 A Liquid/Liquid Separation Technique…using Centrifugal Forces cenapt.pharm.uic.edu Simmler | Friesen | Pauli http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html A centrifuge is used to hold one phase stationary (SP) meanwhile the other phase (mobile) moves through, getting mixed with the SP, then separated throughout the tubing/column
  5. 5. Liquid/Liquid Separation (LLS) Kuhni Extraction Columns Continuous Mixer-Settler Liquid-Liquid Extraction Liquid/Liquid Chromatography A. Martin & R. Synge Centrifugal Countercurrent Separation (CCCS) Gravitational Countercurrent Separation (GCCS) Countercurrent Chromatography (CCC) “hydrodynamic” Centrifugal Partition Chromatography (CPC) “hydrostatic” Droplet Counter Current Chromatogr. Craig Counter Current Distribution Kostanyan Pulsed Rotational Locular Separatory Funnels Countercurrent Separation (CCS) Friesen JB, McAlpine JB, Chen SN, Pauli GF Countercurrent Separation of Natural Products: An Update Journal of Natural Products 78: 1765-1796 (2015) dx.doi.org/10.1021/np501065h Liquid/Liquid Separation Techniques….. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 5
  6. 6. Liquid/Liquid Separation (LLS) Kuhni Extraction Columns Continuous Mixer-Settler Liquid-Liquid Extraction Liquid/Liquid Chromatography A. Martin & R. Synge Centrifugal Countercurrent Separation (CCS) Gravitational Countercurrent Separation (GCS) Countercurrent Chromatography (CCC) “hydrodynamic” Centrifugal Partition Chromatography (CPC) “hydrostatic” Droplet Counter Current Chromatogr. Craig Counter Current Distribution Kostanyan Pulsed Rotational Locular Separatory Funnels Countercurrent Separation (CS) Friesen JB, McAlpine JB, Chen SN, Pauli GF Countercurrent Separation of Natural Products: An Update Journal of Natural Products 78: 1765-1796 (2015) dx.doi.org/10.1021/np501065h Liquid/Liquid Separation Techniques….. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 6
  7. 7. Liquid/Liquid Separation (LLS) Kuhni Extraction Columns Continuous Mixer-Settler Liquid-Liquid Extraction Liquid/Liquid Chromatography A. Martin & R. Synge Centrifugal Countercurrent Separation (CCS) Gravitational Countercurrent Separation (GCS) Countercurrent Chromatography (CCC) “hydrodynamic” Centrifugal Partition Chromatography (CPC) “hydrostatic” Droplet Counter Current Chromatogr. Craig Counter Current Distribution Kostanyan Pulsed Rotational Locular Separatory Funnels Countercurrent Separation (CS) Friesen JB, McAlpine JB, Chen SN, Pauli GF Countercurrent Separation of Natural Products: An Update Journal of Natural Products 78: 1765-1796 (2015) dx.doi.org/10.1021/np501065h Liquid/Liquid Separation Techniques….. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 7
  8. 8. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 8 Examples of “CCC” Hydrodynamic Instruments http://www.dynamicextractions.com/index.html#about1440 Examples of Applications with Guy Harris
  9. 9. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 9 Examples of “CPC” Hydrostatic Instruments https://www.plantaanalytica.com/technology -centrifugal-partition-chromatography.html Examples of Applications with Grégoire Audo
  10. 10. How compounds get separated in CPC/CCC? In HPLC: difference in affinity (column material vs. mobile phase) In CPC/CCC: difference of solubility between two phases The Partition Coefficient K cenapt.pharm.uic.edu Simmler | Friesen | Pauli 10
  11. 11. Partition coefficient (K) = Concentration of analyte in one phase / Concentration of analyte in the other shake flask (partitioning) experiment = 4/4 = 1 = 12/36 = 1/3 K = Cupper/Clower cenapt.pharm.uic.edu Simmler | Friesen | Pauli 11
  12. 12. Separation of Compounds According to their K value K high K = 1 K low stationary phase mobile phase K = conc. stationary/conc. mobile phase cenapt.pharm.uic.edu Simmler | Friesen | Pauli 12
  13. 13. K high K = 1 K low Separation of Compounds According to their K stationary phase mobile phase cenapt.pharm.uic.edu Simmler | Friesen | Pauli 13
  14. 14. Separation of Compounds According to their K K high K = 1 K low K = conc. stationary/conc. mobile phase stationary phase mobile phase cenapt.pharm.uic.edu Simmler | Friesen | Pauli 14
  15. 15. Separation of Compounds According to their K K high K = 1 K low K = conc. stationary/conc. mobile phase stationary phase mobile phase cenapt.pharm.uic.edu Simmler | Friesen | Pauli 15
  16. 16. Separation of Compounds According to their K K high K = 1 K low K = conc. stationary/conc. mobile phase stationary phase mobile phase cenapt.pharm.uic.edu Simmler | Friesen | Pauli 16
  17. 17. What is a biphasic solvent system in CCS? How to choose an appropriate solvent system ? Evaluate the overall distribution of compounds in the UP and LP of a solvent system (K) 17cenapt.pharm.uic.edu Simmler | Friesen | Pauli
  18. 18. Biphasic Solvent System = Mixture of multiple solvents that form two immiscible phases Usually more than 2 solvents ( generally 3-4) are utilized in CCS to increase the selectivity towards the compounds of interest. Examples: cenapt.pharm.uic.edu Simmler | Friesen | Pauli 18 1 hexane : acetonitrile : methanol 10:5:5 HAcM 2 hexane : methyl t-butyl ether : acetonitrile : water 8:2:8:2 HterAcWat 3 hexane : ethyl acetate : methanol: water 5:5:5:5 HEMWat 4 chloroform : methanol : water 10:7:3 ChMWat 5 methyl t-butyl methyl ether : acetonitrile : water 4:6:10 terAcWat 6 ethyl acetate : 1-butanol : water 4:6:10 EBuWat 7 hexane : methyl t-butyl ether : acetonitrile 10:1:10 HterAc 8 dichloromethane: ethyl acetate : methanol : water 5:5:5:5 DEMWat 9 hexane : methyl t-butyl ether : methanol : water 5:5:5:5 HterMWat Scout Solvent Systems
  19. 19. Diversity of Biphasic Solvent Systems Alcohol/Ionic Aqueous or Acetonitrile/Ionic Aqueous Aqueous Two Phase Solvent Systems (ATPS) e.g. Polyethylene Glycol/Buffered Aqueous Organic/Organic or “non-aqueous” e.g. Heptane/Methanol Organic/Aqueous e.g. Hexanes/Ethyl Acetate/Methanol/Water cenapt.pharm.uic.edu Simmler | Friesen | Pauli
  20. 20. Where to Start? ….Which Biphasic Solvent System? ….What K? Literature Measure K G.U.E.S.S Predict (in silico) TLC based Scout systems Reported systems LC/GC/ NMR… TLC Structure + solvents Shake-flask Real partitioning 1. 2. Liu Y, Friesen JB, McAlpine JB, Pauli GF Solvent System Selection Strategies in Countercurrent Separation Planta Medica 81: 1582-1591 (2015) Brent Friesen, J. Pauli, Guido F. “G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC” Journal of Liquid Chromatography & Related Technologies 28 (17): 2777-2806 (2005) Laura Tyler poster #93
  21. 21. Where to Start? ….. Which Solvent System? ….What K? Literature Measure K G.U.E.S.S Predict (in silico) TLC based Scout systems Reported systems LC/GC/ NMR… TLC Structure + solvents Shake-flask Real partitioning 1. 2. Liu Y, Friesen JB, McAlpine JB, Pauli GF Solvent System Selection Strategies in Countercurrent Separation Planta Medica 81: 1582-1591 (2015) Brent Friesen, J. Pauli, Guido F. G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC Journal of Liquid Chromatography & Related Technologies 28 (17): 2777-2806 (2005) COSMO-RS model
  22. 22. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 22 Where to Start ? …. Which Solvent System? The different organic/aqueous solvent system families Abbreviations Solvent mixtures HEMWat Hexanes/Ethyl Acetate/Methanol/Water ChMWat Chloroform/Methanol/Water EBuWat Ethyl Acetate/Butanol/Water terAcWat Methyl t-Butyl Ether/Acetonitrile/Water 1. Perform Shake-flask experiment with the portal mixture of each solvent system family 2. Observe/measure the distribution of your compounds in UP and LP (TLC, HPLC, NMR) More polar, glycosidated cpds Workhorse
  23. 23. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 23 Working with Solvent System Families… EBuWat & terAcWat Friesen, J Brent, and Guido F Pauli. Rational Development of Solvent System Families in Countercurrent Chromatography. Journal of Chromatography A 1151, no. 1–2 (June 1, 2007): 51–59. Portal = first solvent composition to try in the considered family
  24. 24. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 24 Working with System Families…. HEMWat organic/organic modifier/aqueous modifier/aqueous + versatile and tunable • heptane, or pet. ether, or limonene for hexanes • ethanol for methanol + good CCC/CPC performance (high Sf values) Hexane/ Ethyl Acetate / Methanol/ Water = HEMWat (or ARIZONA) family of solvent systems Mcalpine, James B, J Brent Friesen, and Guido F Pauli. Natural Products Isolation. Edited by Satyajit D. Sarker and Lutfun Nahar. Vol. 864. Methods in Molecular Biology. Totowa, NJ: Humana Press, 2012. Portal: working very well for first step fractionation of crude extracts! Laura Tyler poster #93
  25. 25. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 25 James B McAlpine, J Brent Friesen, and Guido F Pauli. Natural Products Isolation. Edited by Satyajit D. Sarker and Lutfun Nahar. Vol. 864. Methods in Molecular Biology. Totowa, NJ: Humana Press, 2012. Often modified by adding Hexanes → HChMWat Koichi Inouie et al. “Purification of Curcumin , Demethoxycurcumin , and Bisdemethoxycurcumin by High-Speed Countercurrent Chromatography,” Journal of Agricultural and Food Chemistry 2008, 9328–36. Purification of curcuminoids from 25 mg of turmeric powder Curcumin K= 0.68 Bidesmethoxycurcumin K = 1.64 Desmethoxycurcumin K = 1.03 Working with Solvent System Families... ChMWat Portal
  26. 26. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 26 Basic Principle of a Shake-Flask Experiment to (1) measure K (2) observe the distribution of compounds in both phases Same volume 1. Mix 2-phase SS 2. Add sample to vial 3. Add equal amounts of upper and lower phase 4. Shake it up! 5. Separate upper and lower phase 6. Analysis of upper and lower phase Same volume
  27. 27. Shake-Flask and Solvent System Selection cenapt.pharm.uic.edu Simmler | Friesen | Pauli 27 HEMWat 0 5/5/5/5 TerAcWat-1 4/6/10 ChMWat +4 10:7:3 Turmeric Extract UP LP 25 mg 6 mL 6 mL Comparison of different portal solvent systems
  28. 28. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 28 HEMWat 5/5/5/5 HEEWat 5/5/5/5 TerAcWat 4/6/10 ChMWat 10/7/3 HChMWat 5/10/7/3 HTerAcWat 2/4/6/10 Better solubility Push cpds towards aqueous phase Speed-up phase separation Push cpds towards aqueous phase Speed-uo phase separation
  29. 29. Compound distribution in the UP and LP cenapt.pharm.uic.edu Simmler | Friesen | Pauli 29 Spot the same volume of UP and LP HEMWat 5/5/5/5 HEEWat 5/5/5/5 TerAcWat 4/6/10 ChMWat 10/7/3 HChMWat 5/10/7/3 HTerAcWat 2/4/6/10 HEMWat 5/5/5/5 HEEWat 5/5/5/5 TerAcWat 4/6/10 ChMWat 10/7/3 HChMWat 5/10/7/3 HTerAcWat 2/4/6/10 365 nmVisible UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP
  30. 30. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 30 M 5444 E 5444 M 6464 E 6464 M 5444 E 5444 M 6464 E 6464 M 5444 E 5444 M 6464 E 6464 UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP UP LP HEMWat Solvent System: Replacing MeOH by EtOH Checking the overall distribution of your compounds in both phases by TLC ( same volume spotted ~ 10 µL) Reduces overall emulsion tendency, favors a clear phase separation HEMWat 5:4:4:4 HEEtWat 5:4:4:4 CPC for chlorophyll removal? Seon-Beom Kim’s poster # 240
  31. 31. Liu Y, Friesen JB, McAlpine JB, Pauli GF Solvent System Selection Strategies in Countercurrent Separation Planta Medica 81: 1582-1591 (2015) Where to Start? ….. Which Solvent System? ….What K? Literature Measure K G.U.E.S.S Predict (in silico) Brent Friesen, J. Pauli, Guido F. G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC Journal of Liquid Chromatography & Related Technologies 28 (17): 2777-2806 (2005) TLC based Scout systems Reported systems LC/GC/ NMR… TLC Structure + solvents Shake-flask Real partitioning 1. 2. cenapt.pharm.uic.edu Simmler | Friesen | Pauli
  32. 32. Basic Principle of G.U.E.S.S. Vol. K = 1 K = 0.25 K = 4 Rf = 0.5 Rf = 0.6 Rf = 0.4 sweet spot TLC Generally Useful Estimation of Solvent Systems combines the convenience of TLC with the separation power of HSCCC. CCC TLC elution with the UP of your Solvent System cenapt.pharm.uic.edu Simmler | Friesen | Pauli Brent Friesen, J. Pauli, Guido F. “G.U.E.S.S.—A Generally Useful Estimate of Solvent Systems for CCC” Journal of Liquid Chromatography & Related Technologies 28 (17): 2777-2806 (2005)
  33. 33. • Start with 2 immiscible solvents and add one or more modifiers • Check the overall distribution of your compounds in both phases. Chloroform/Water Chloroform/Methanol/Water (ChMWat) Hexane/Water Hexane/Methanol/Water Hexane/ Ethyl Acetate /Water Hexane/ Ethyl Acetate /Methanol/Water (HEMWat) Be Bold…..Create Your Solvent System Mixology cenapt.pharm.uic.edu Simmler | Friesen | Pauli Dr. Malca’s poster # 204
  34. 34. Question 1 – Why K? 1. Knowing the K values of your compounds helps you calculate their retention times during your CCS. 2. Knowing the K values helps you to compare different CCS experiments, on different instruments. Question 2 – Really?? 1. If you just want to fractionate a crude extract, you don’t need to determine the K of your compounds. 2. In this case you can run a shake-flask experiment and simply observe the overall distribution of compounds between LP and UP to choose your SS. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 34 1. Why do I Need to Determine the K Value of Compounds? 2. Is it Always Necessary? Dr. Zhou poster # 314, Dr. Malca’s poster # 204, Dr Tang’s poster # 117
  35. 35. Some Key Parameters when running a CCS  Elution mode  K vs. volume of retention  Sf and column volume  Rotation speed & flow rate cenapt.pharm.uic.edu Simmler | Friesen | Pauli 35
  36. 36. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 36 Parameters in CCS : Direction of elution Choosing which phase is your stationary phase Upper phase = Stationary phase Reversed phase chromatography Lower phase = Stationary phase Normal phase chromatography Lower phase = mobile HEAD TAIL DESCENDING Upper phase = mobile Get mixed Goes back up ASCENDING
  37. 37. Relation between K & vol. of retention in your chromatogram cenapt.pharm.uic.edu Simmler | Friesen | Pauli 37 Vret Retention Volume VSP Stationary Phase (SP) Volume in the column VMP Mobile Phase (MP) Volume in the column K Partition Coefficient Vret = K X VSP + VMP You can exactly determine in which test tubes are your compounds of interest without hyphenated detection! How to determine VSP and VMP
  38. 38. cenapt.pharm.uic.edu Simmler | Friesen | Pauli 38 column Stationary phase (SP)1. Fill the column with SP 2. Apply centrifugal forces = rotation (speed in rpm) 3. Introduce the MP at chosen flow rate 4. Equilibrium between SP/MP When MP comes out of the coil. SF can be calculated. SPMP Vout Vcolumn VSP VMP OUTIN Parameters in CCS : The stationary phase volume ratio Sf Sf = VSP/Vcolumn Sf = (Vcolumn –Vout)/Vcolumn To determine VSP and VMP 10 20 30 40 50 60 70 80 90 100
  39. 39. Now you have your solvent system… You can start your CCS…. 1. Dissolve your sample in mixture of UP/LP (generally 50%) • Everything should be in solution, no particles • You may want to filter your sample • Write down – mass of sample/volume of solvent 2. Fill your CPC/CCC instrument with the phase you chose as stationary • 2 column volumes minimum, • Slow rotation of the column(s) 3. Start rotating your instrument at the desired speed • Ex: 800 rpm for CCC, 2500 rpm for CPC 4. Set up your mode of elution (direction of the column rotation) • Stationary phase = organic (UP) direction of flow = descending or “head to tail” 5. Start pumping your mobile phase into the instrument at the desired flow rate • Ex: 1.5-2 mL/min in CCC, 25 mL/min in CPC 6. Wait for the “equilibrium” to be reached (or not, your choice).. • to determine how much of the stationary phase remains in the column (Sf) 7. Inject your sample ….beware: do not inject air bubbles! 8. Collect your fractions! cenapt.pharm.uic.edu Simmler | Friesen | Pauli 39 Degas your solvent before use! Before loading the sample, fill the loop with your MP!
  40. 40. The ASP organizing committee, particularly Laura Stoll NCCIH and ODS at NIH (U41 AT008706) ACKNOWLEDGEMENTS
  41. 41. REPOSITORY REPORTING CCS PARAMETERS EXAMPLE OF APPLICATIONS https://cenapt.pharm.uic.edu 41
  42. 42. 42 Reported Parameters for Reproducibility With a CPC instrument With an HSCCC instrumentDuration = 1 hour max Duration = 5 hours 25 mg 780 rpm 1mL/min 4.24 g2500 rpm 25 mL/min INOUE, KOICHI, et al. Journal of Agricultural and Food Chemistry 56, no. 20 (2008): 9328–36.Simmler, C., et al. Fitoterapia 121 (2017): 6-15. Crude extract fractionation cenapt.pharm.uic.edu Simmler | Friesen | Pauli
  43. 43. Countercurrent Separation as a first step for the reproducible fractionation of crude botanical extract CE Fr1 GI0345 01 Fr2 GI0345 02 Fr3 GI0345 03 Fr4 GI0345 04 Fr5 GI0345 05 Fr6 GI0345 06 Fr7 GI0345 07 Fr8 GI0345 08 Fr9 GI0345 09 Fr10 GI045 10 Fr11 Gi0345 11 Glycyrrhiza inflata Crude extract (3x 4g injected) HEMWat 0 3x 50 min max ½ prepHPLC ½ prepHPLC Sf= 83% 25 mL/min, 2500 rpm Reverse mode K= 1.02 Precipitation: 90.9% w/w 13 mL fractions Simmler/Lankin/Nikolić/van Breemen/Pauli Fitoterapia 121 (2017). doi:10.1016/j.fitote.2017.06.017 new new Licochalcone A FRACTIONATION PURIFICATION cenapt.pharm.uic.edu Simmler | Friesen | Pauli
  44. 44. Crudeextract Fr1 GI0345 01 Fr2 GI0345 02 Fr3 GI0345 03 Fr4 GI0345 04 Fr5 GI0345 05 Fr6 GI0345 06 Fr7 GI0345 07 Fr8 GI0345 08 Fr9 GI0345 09 Fr10 GI045 10 Fr11 Gi0345 11 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 Crude extract + - 51.6% w/w 8.2% w/w Mass balance recovery : 90.8% w/w Licochalcone A UHPLC-UV profiles LicA LicA Countercurrent Separation as a first step for the reproducible fractionation of crude botanical extract cenapt.pharm.uic.edu Simmler | Friesen | Pauli

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