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Phase modifications

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What are the different parameters to consider for the modification of a chosen solvent system to be utilized in countercurrent chromatography.

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Phase modifications

  1. 1. CounterCurrent Separation Theory and practice §Minimal sample preparation (direct chromatography of crude extracts) §High mass – High resolution §No sample loss (support-free chromatography) §Reproducibility (scale-up or scale down) §Flexibility §Mild conditions for sensitive molecules
  2. 2. Modification of Solvent Systems Phase modification before (column) pre-equilibration - Inorganic salts - DMSO / Ionic Liquids - Acid and/or Base - Ion-Pairing Factors - Salts that Create Hydro-alcoholic Systems
  3. 3. Modification of Solvent Systems Phase modification before (column) pre-equilibration - Inorganic salts
  4. 4. addition of salts Salting-out gradients in centrifugal partition chromatography for the isolation of chlorogenic acids from green coffee Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4245-4251 Roman R. Romero-González, Robert Verpoorte Fig. 2. Salting effect of selected salts on chlorogenic acid, 5-CQA, in an ethyl acetate-aqueous salt solution (pH 2.5) biphasic system. Distribution constants are defined as the concentration ratio between the most lypophilic phase and the most hydrophilic one. Maximum salt concentrations are determined by their solubility limits.
  5. 5. Addition of Salts Partition efficiencies of newly fabricated universal high-speed counter-current chromatograph for separation of two different types of sugar derivatives with organic-aqueous two-phase solvent systems Kazufusa Shinomiya J Chromatogr A 1322:74-80. 2013
  6. 6. Partition efficiencies of newly fabricated universal high-speed counter-current chromatograph for separation of two different types of sugar derivatives with organic-aqueous two-phase solvent systems Kazufusa Shinomiya J Chromatogr A 1322:74-80. 2013 Addition of Salts
  7. 7. Anal Bioanal Chem. 2011 Jul;400(10):3615-23. doi: 10.1007/s00216-011-4995-2. High-speed counter-current chromatographic separation of phytosterols. Schröder M1, Vetter W. Addition of Salts
  8. 8. Modification of Solvent Systems Phase modification before (column) pre-equilibration - DMSO / Ionic Liquids
  9. 9. DMSO additive Figure 1. HPLC fractogram of a CPC purification of protected exenatide (2 g); solvent system: Heptane/THF/MeCN/DMSO/W, 19.1:36.2:14:17.6:13 v/v; descending mode,flow rate: 4 mL/min plus 5% stationary phase co-current; rotation speed: 2000 rpm; stationary phase retention: 74%; UV detection: = 215 nm; RRT: relative retention time in HPLC. HepTetAcSoWat J Chromatogr A. 2014 Apr 11;1337:155-61. doi: 10.1016/j.chroma.2014.02.052. Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography. Amarouche N, Giraud M, Forni L, Butte A, Edwards F, Borie N, Renault JH.
  10. 10. Methyl furan and N-methyl pyrrolidone Figure 2. HPLC fractogram of a CPC purification of 8-mer protected peptide (1 g); solvent system: Heptane/Me-THF/NMP/water, 7:50:23:20, v/v; descending mode, flowrate: 8 mL/min; extrusion step at 31 min, rotation speed: 1400 rpm; stationary phase retention: 72%; UV detection: = 215 nm; RRT: relative retention time in HPLC. Hep/Me-THF/NMP/Wat J Chromatogr A. 2014 Apr 11;1337:155-61. doi: 10.1016/j.chroma.2014.02.052. Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography. Amarouche N, Giraud M, Forni L, Butte A, Edwards F, Borie N, Renault JH.
  11. 11. Modification of Solvent Systems Phase modification before (column) pre-equilibration - Acid and/or Base
  12. 12. Influence of pH on K Fig. 1. Effect of the total benzoic acid concentration (top) and aqueous phase pH (bottom) on the benzoic acid distribution ratio, KC. Heptane to buffer volume ratio is one to one. The inset in the top figure shows the linear change of the benzoic acid distribution ratio at pH 7. The inset in the bottom figure shows the benzoic acid adsorption isotherms (concentration in the aqueous phase versus concentration in the heptane phase) at different mobile phase pH values (1, 3, 4.2, 5 and 6). Journal of Chromatography A, 1218 (2011) 6024–6030 Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid Alain Berthod∗, Nazim Mekaoui
  13. 13. Fig. 3. Retention of benzoic acid at different pH values. Liquid system Composition 2, heptane/butanol/buffer 200/20/380 (Table 2). Mobile aqueous lower phase at 0.8 mL/min in the descending head-to-tail direction. Hydrodynamic CCC column Milli, 18 mL, rotor rotation speed 2000 rpm, 30 ◦C. Benzoic acid injected mass: 0.5 mg, injected volume: 0.5 mL. UV detection at 254 nm. Aqueous buffer pHs are: A – 6.96; B – 4.61; C – 3.62; and D – 2.19. Journal of Chromatography A, 1218 (2011) 6024–6030 Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid Alain Berthod∗, Nazim Mekaoui Influence of pH on K
  14. 14. Influence of pH on K Countercurrent purification of the tetrahydro iso-α acids derived from Humulus lupulus L. JOURNAL OF SEPARATION SCIENCE Volume 33, Issue 17-18, September 2010, Pages: 2828–2832, Clinton J. Dahlberg, James S. Traub, Matthew L. Tripp, Jeffrey S. Bland and Brian J. Carroll
  15. 15. Influence of pH on K Countercurrent purification of the tetrahydro iso-α acids derived from Humulus lupulus L. JOURNAL OF SEPARATION SCIENCE Volume 33, Issue 17-18, September 2010, Pages: 2828–2832, Clinton J. Dahlberg, James S. Traub, Matthew L. Tripp, Jeffrey S. Bland and Brian J. Carroll Figure 2. Overlay of the UV chromatogram from the CS experiment (entry 3, Table 1, 254 nm) with a reconstructed CS chromatogram (254 nm) from the HPLC analysis of collected 28 mL fractions. HEMWat (acetate pH 5.3) 7:3:5:5
  16. 16. Influence of pH on K Isolation of bitter acids from hops (Humulus lupulus L.) using countercurrent chromatography JOURNAL OF SEPARATION SCIENCE Volume 35, Issue 9, May 2012, Pages: 1183–1189, Clinton J. Dahlberg, Guy Harris, Jan Urban, Matthew L. Tripp, Jeffrey S. Bland and Brian J. Carroll Figure 5. CCC chromatogram (254 nm) using a HEMWat 5:5:5:5, 250 mM NH4Ac; pH 5.1 solvent system for the purification of the RIAA diastereomers produced by NaBH4 reduction of the C6 keto in cis n-IAA (isohumulone).
  17. 17. Modification of Solvent Systems Phase modification before (column) pre-equilibration - Ion-Pairing Factors
  18. 18. Ion Pairs Fig. 2. HSCCC-chromatogram of C18-enriched pigment extract (1677 mg) I. lindeniiVan Houtte leaves. HSCCC conditions: flow rate: 3.0 mL/min; ‘head-to-tail mode’; biphasic solvent system TBME–1-BuOH– ACN–H2O (0.7% HFBA) 1:3:1:5 (v/v/v/v).Below, HPLC retention times of the separated major betacyanins detected in each HSCCC fraction. Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent chromatography Journal of Chromatography A, Volume 1344, 30 May 2014, Pages 42-50 Gerold Jerz, Nadine Gebers, Dominika Szot, Maciej Szaleniec, Peter Winterhalter, Slawomir Wybraniec https://nl.wikipedia.org/wiki/Betanine#/media/File:Betanin.svg
  19. 19. Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent chromatography Journal of Chromatography A, Volume 1344, 30 May 2014, Pages 42-50 Gerold Jerz, Nadine Gebers, Dominika Szot, Maciej Szaleniec, Peter Winterhalter, Slawomir Wybraniec Target-guided separation of Bougainvillea glabra betacyanins by direct coupling of preparative ion-pair high-speed countercurrent chromatography and electrospray ionization mass-spectrometry Journal of Chromatography A, Volume 1217, Issue 27, 2 July 2010, Pages 4544-4554 Gerold Jerz, Sławomir Wybraniec, Nadine Gebers, Peter Winterhalter terBuAcWat (HFBA) 1:3:1:5 terBuAcWat (TFA) 2:2:1:5 Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography Journal of Chromatography A, Volume 1190, Issues 1–2, 9 May 2008, Pages 63-73 Gerold Jerz, Tanja Skotzki, Kathrin Fiege, Peter Winterhalter, Sławomir Wybraniec BuAcWat (TFA) 5:1:6 (0.7%) Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography Journal of Chromatography A, Volume 1216, Issue 41, 9 October 2009, Pages 6890-6899 Sławomir Wybraniec, Paweł Stalica, Gerold Jerz, Bettina Klose, Nadine Gebers, Peter Winterhalter, Aneta Spórna, Maciej Szaleniec, Yosef Mizrahi BuAcWat (TFA) 5:1:6 (0.7%) terBuAcWat (TFA/HFBA) 2:2:1:5 (0.7%) New solvent systems for gradient counter-current chromatography in separation of betanin and its derivatives from processed Beta vulgaris L. juice. Journal of Chromatography A, Volume 1380, 6 February 2015, Pages 29-37 Aneta Spórna-Kucab, Ian Garrard, Svetlana Ignatova, Sławomir Wybraniec BuEtWat/Aq. NaCl/Aq. H3PO4 1300:200-1000:1300:700:2.5-10 Ion-pair high-speed countercurrent chromatography in fractionation of a high-molecular weight variation of acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra Journal of Chromatography B, Volume 878, Issues 5–6, 15 February 2010, Pages 538-550 Sławomir Wybraniec, Gerold Jerz, Nadine Gebers, Peter Winterhalter BuAcWat (TFA) 5:1:6 (0.7%) terBuAcWat (TFA) 2:2:1:5 (0.7%) Fig. 3. IP–HSCCC chromatogram of directly recovered crude pigment extracts (755 mg) of violet B. glabra bracts in the solvent system terBuAcWat 2:2:1:5 using 0.7% TFA. HSCCC conditions: flow rate: 3.0 mL/min; CCC-operation: head-to-tail mode; velocity: 850 rpm and detection wavelength 540 nm. Ion Pairs
  20. 20. Modification of Solvent Systems Phase modification before (column) pre-equilibration - Salts that Create Hydro-alcoholic Systems
  21. 21. Hydroalcoholic Systems Journal of Liquid Chromatography & Related Technologies Volume 29, Issue 5, 2006 Countercurrent Chromatographic Separation of Biotic Compounds with Extremely Hydrophilic Organic-Aqueous Two-Phase Solvent Systems and Organic-Aqueous Three-Phase Solvent Systems DOI:10.1080/10826070500509298 Kazufusa Shinomiya & Yoichiro Ito Figure 1. CCC Chromatogram of proteins obtained by the X-axis CPC using ethanol/ aqueous 2M ammonium sulfate (3 : 5) with the upper phase mobile. Experimental conditions: apparatus: X-axis CPC equipped with a pair of eccentric coil assemblies with 1mm ID and a total capacity of 26.5 mL; sample: 5mg each of human serum albumin and lysozyme; solvent system: ethanol/aqueous 2M ammonium sulfate (3 : 5); mobile phase: upper phase; flow rate: 0.4mL/min; revolution: 800 rpm. SF ¼ solvent front.
  22. 22. Hydroalcoholic Systems Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system Journal of Chromatography A, Volume 1217, Issue 20, 14 May 2010, Pages 3457-3460 Yoichi Shibusawa, Atsushi Shoji, Chihiro Suzuka, Akio Yanagida, Yoichiro Ito Fig. 3. Separation of eight nucleic acid constituents by HSCCC using hydrophilic solvent system. Dimension of PTFE multilayer coiled column: 1.0mm I.D.×50m (40 ml capacity); solvent system: 1-propanol/800mM potassium phosphate buffer (pH 7.4) (1:1, v/v); stationary phase: upper phase; mobile phase: lower phase; flowrate: 0.5 ml/min; revolution: 1000 rpm; UP = the starting point at which the upper phase was used as the mobile phase. For the components’ identification, see Fig. 1.
  23. 23. Modification of Solvent Systems Phase modification before (column) pre-equilibration - Hydrophobic Compounds
  24. 24. Hydrophobic Compounds Fig. 1. Partition coefficients of four critical pairs of fatty acids versus the percentage of water in the mobile phase in HepMWat. 1 = C16:3; 2 = C18:4; 3 = C20:5; 4 = C22:6. Counter-current chromatographic separation of polyunsaturated fatty acids Journal of Chromatography A, Volume 704, Issue 1, 2 June 1995, Pages 211-216 Olivier Bousquet, François Le Goffic
  25. 25. Hydrophobic Compounds (a) methyl p-hydroxybenzoate, (b) ethyl p-hydroxybenzoate, (c) ethyl m-hydroxybenzoate (d) isopropyl p- hydroxybenzoate, (e) propylp-hydroxybenzoate, (f) isobutyl p-hydroxybenzoate and (g) butyl p- hydroxybenzoate HCyterMWat 5:0:2:5:3 HCyterMWat 4:1:2:5:3 HCyterMWat 3:2:2:5:3 HCyterMWat 2:3:2:5:3 HCyterMWat 1:4:2:5:3 HCyterMWat 0:5:2:5:3 J Chromatogr A. 2014 May 16;1342:54-62. doi: 10.1016/j.chroma.2014.03.050. Solvent systems with n-hexane and/or cyclohexane in countercurrent chromatography-- Physico-chemical parameters and their impact on the separation of alkyl hydroxybenzoates. Englert M1, Vetter W2.
  26. 26. Hydrophobic Compounds Fig. 6. CCC-UV–vis chromatogram (450 nm) of the separation of crude carrot extract with dual-mode utilization (mobile phase and elution mode reversal). Conditions:CCC-1000 with three serially connected multilayer coils (1.6 mm i.d. PTFE tubing)and a total column capacity of 325 mL and 10 mL sample loop; rotation speed:1010 ± 10 rpm; solvent system: n- H/benzotrifluoride/Ac (10:3.5:6.5,v/v/v); sample size: 100.2 mg; separation: dual mode CCC separation with tail-to-head mode for 140 min at a flow rate of 2 mL/min and head-to- tail elution for 60 min at a flow rate of 4 mL/min. J Chromatogr A. 2015 Apr 3;1388:119-25. doi: 10.1016/j.chroma.2015.02.020. Isolation of β-carotene, α-carotene and lutein from carrots by countercurrent chromatography with the solvent system modifier benzotrifluoride. Englert M, Hammann S, Vetter W.

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