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solvent system formulation

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Parameters to consider for the formulation of bi(tri)phasic solvent systems to be utilized in liquid/liquid countercurrent chromatography.

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solvent system formulation

  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. How does a separatory funnel work? Liquid- Liquid Extraction 2. Add compound mixture (solutes) 3. Shake (vigorous mixing of phases) 4. Allow phases to separate (settling) 5. Physically separate layers 1. Formulate biphasic solvent mixture (system) Solvent System = SS Phase System 6. Analyze layers for composition
  3. 3. Liquid- Liquid Extraction Simple biphasic solvent systems are created by mixing two immiscible liquids. Organic/Aqueous liquid/liquid separation
  4. 4. Biphasic solvent systems may have 2 or more solvents. Liquid- Liquid Extraction A common way of formulating solvent system is to start with 2 immiscible solvents and add one or more modifiers. Chloroform/Water Chloroform/Methanol/Water (ChMWat) Hexane/Water Hexane/Methanol/Water Hexane/ Ethyl Acetate /Water Hexane/ Ethyl Acetate /Methanol/Water HEMWat
  5. 5. Survey Results 23 133 223 10 3 2 3 4 5 6 number of solvents number of solvents per SS Friesen 2015 J Nat Prod v. 78 p. 1764 Liquid- Liquid Extraction
  6. 6. Ternary Phase Diagrams (for three-component solvent systems) Liquid- Liquid Extraction
  7. 7. Solvent System Characterization Berthod, A. (2002). Countercurrent Chromatography: The support-free liquid stationary phase. Wilson & Wilson's Comprehensive Analytical Chemistry Vol. 38. Boston: Elsevier Science Ltd. pp. 1–397. ISBN 978-0-444-50737-2.
  8. 8. Fig. 3. (a) Chemical structure of benzotrifluoride and (b) ternary phase diagram of n- hexane/benzotrifluoride/acetonitrile with two-phase region at 22◦C shown in grey shading. Volumetric solvent composition for the CCC separation is represented as ared triangle (50% n-hexane, 17.5% benzotrifluoride, 32.5% acetonitrile, 10:3.5:6.5).The tie-lines indicate that benzotrifluoride partitioned evenly between n- hexane and acetonitrile. The composition of the upper and lower phases varied extensively with the amount of benzotrifluoride in the system 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. Solvent System Characterization
  9. 9. Liquid- Liquid Extraction Biphasic solvent systems created by mixing two or more immiscible liquids. Alcohol/Ionic Aqueous or Acetonitrile/Ionic Aqueous Aqueous Two Phase Solvent Systems (ATPS) Polyethylene Glycol/Buffered Aqueous Organic/Organic or “non-aqueous” (heptane/methanol) Organic/Aqueous (ChMWat, HEMWat, etc…)
  10. 10. Unified Solvent Notation Petroleum ether Pet DMSO So Hexane H Acetonitrile Ac Heptane Hep Isopropyl alcohol Iso Cyclohexane Cy n-propanol Pro Toluene Tol n-butanol Bu Methyl tert-butylether ter Acetic acid Aa THF Tet Ethanol Et Diethyl ether De Methanol M Ethyl acetate E Water Wat Methyl acetate Me Chloroform Ch Dichloromethane Di Acetone At 1. Solvent Abbreviations Used Define Countercurrent Separation Solvent Systems
  11. 11. Unified Solvent System Notation 2. Order of polarity: least polar to most polar à HEMWat, ChMWat, terBuAcWat 4. Whole numbers à HEMWat 2:3:4:5 4a. No numbers needed for binary solvent systems 5b. Lowest common denominator? 5:5:5:5 or 1:1:1:1 6. pre-equilibration additives/modifiers in parentheses à terBuAcWat (0.7% TFA) 6a. What does concentration mean? Concentration in a single component or overall mixture? 3. Volume ratios à HEMWat 2:3:4:5 6b. Modifier or solvent component? acetic acid, ionic liquids, 1. Solvent abbreviations 1a. Write out new or rare solvents 7. pH-zone refining and Ion-exchange (retainer - eluter)
  12. 12. HEMWat http://2.bp.blogspot.com/-4jotcEHY_Iw/Tu7oPvHVX9I/AAAAAAAAAyk/fsircQR9n3A/s1600/workhorse_discing.jpg
  13. 13. The HEMWat SS Family Biphasic solvent systems created by mixing two or more immiscible liquids. organic/organic modifier/aqueous modifier/aqueous + low-cost + versatile and tunable (heptane or pet ether or limonene for hexane) (ethanol for methanol) + good CCC/CPC performance (high Sf values) Hexane/ Ethyl Acetate /Methanol/Water = HEMWat - environmental impact / health concerns - stability - what about pH, shouldn’t we buffer this? + create solvent system families by systematic modification
  14. 14. The HEMWat SS Family HEMWat solvent composition 0% 25% 50% 75% 100% +8 +7 +6 +5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5 -6 -7 -8 HEMWat number Hexane EtOAc MeOH Water Friesen B & Pauli GF G.U.E.S.S. to make Generally Useful Estimations of Solvent Systems in CCC. Journal of Liquid Chromatography & Relat. Technologies 28: 2777-2806 (2005) dx.doi.org/10.1080/10826070500225234
  15. 15. The HEMWat SS Family Oka F, Oka H, Ito Y (1991) J Chromatogr A 538:99–105
  16. 16. Anal Bioanal Chem. 2005 Sep;383(2):327-340. Alkane effect in the Arizona liquid systems used in countercurrent chromatography. Berthod A1, Hassoun M, Ruiz-Angel MJ. 10.1007/s00216-005-0016-7 The HEMWat SS Family Renault JH, Nuzillard JM, Intes O, Maciuk A (2002) In: Berthod A (ed) Countercurrent chromatography, the support free liquid stationary phase (Comprehensive analytical chemistry, vol. 38). Elsevier, Amsterdam, pp 49–83 The HEMWatSS family
  17. 17. The HEMWat SS Family Dubant S, Mathews B, Higginson P, Crook R, Snowden M, Mitchell J. Practical solvent system selection for counter-current separation of pharmaceutical compounds. J Chromatogr A 2008; 1207: 190–192 The HEMWat SS family
  18. 18. A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural productsOriginal Research Article Journal of Chromatography A, Volume 1400, 26 June 2015, Pages 27-39 Junling Liang, Jie Meng, Dingfang Wu, Mengzhe Guo, Shihua Wu doi:10.1016/j.chroma.2015.04.043 The HEMWat SS Family 8 x 8 = 64 possibilitiesThe HEMWat SS family
  19. 19. The HEMWat SS Family J Chromatogr A. 2014 Nov 14;1368:116-24. doi: 10.1016/j.chroma.2014.09.064. Folding fan mode counter-current chromatography offers fast blind screening for drug discovery. Case study: finding anti-enterovirus 71 agents from Anemarrhena asphodeloides. Liu M, Tao L, Chau SL, Wu R, Zhang H, Yang Y, Yang D, Bian Z, Lu A, Han Q, Xu H8.
  20. 20. 461 261 232 172 104 42 35 33 24 23 22 18 8 5 3 2 2 1 water ethyl acetate methanol hexane n-butanol acetonitrile ethanol chloroform pet ether heptane methyl t-butyl ether isopropanol dichloromethane acetone ethyl ether cyclohexane carbon tetrachloride THF Survey Results Friesen2015_JNP_78_1764_countercurrent Solvents used in Biphasic SSs 97% had water 35% HEMWat 16% similar to HEMWat The HEMWat SS Family
  21. 21. Solvent System Properties http://us.hola.com/imagenes/viajes/2011071553613/chicago-moderno-estados-unidos/0-181-586/a_009GenSM-a.jpg
  22. 22. Solvent System Properties Polarity Selectivity Friesen, J. B.; Ahmed, S.; Pauli, G. F. Qualitative and quantitative evaluation of solvent systems for countercurrent separation. Journal of Chromatography A 2015, 1377, 55–63 DOI: 10.1016/j.chroma.2014.11.085. Will my chromatography method position the target compounds with a reasonable VR (tR)? Not in void volume. Not highly retained on the column Can molecules with similar polarity be separated in that region with a reasonable VR? match between solvent system and solute (polarity scales such as LogPo/w , Reichardt’s Dye scale, and eluotropic sequences )
  23. 23. Polarity 1. (Linear combination of) Eluotropic sequence parameters 2. Solvatochromic (Reichardt’s) Dye 3. GUESSmix method 4. Phase Metering Apparatus
  24. 24. (Linear combination of) Eluotropic sequence parameters Journal of Chromatography A Volume 1393, 8 May 2015, Pages 47–56 Systematic and practical solvent system selection strategy based on the nonrandom two-liquid segment activity coefficient model for real-life counter-current chromatography separation Da-Bing Ren, Lun-Zhao Yi, Yan-Hua Qin, Yong-Huan Yun, Bai-Chuan Deng, Hong-Mei Lu, Xiao- Qing Chen, Yi-Zeng Liang,
  25. 25. Journal of Chromatography A Volume 1393, 8 May 2015, Pages 47–56 Systematic and practical solvent system selection strategy based on the nonrandom two-liquid segment activity coefficient model for real-life counter-current chromatography separation Da-Bing Ren, Lun-Zhao Yi, Yan-Hua Qin, Yong-Huan Yun, Bai-Chuan Deng, Hong-Mei Lu, Xiao- Qing Chen, Yi-Zeng Liang, (Linear combination of) Eluotropic sequence parameters
  26. 26. Solvent X Y- Y+ Z acetonitrile 0.018 0.131 0.883 0.000 chloroform, 0.393 0.0 0.167 0.000 ethyl acetate 0.3339 0.058 0.441 0.000 ethanol 0.251 0.030 0.000 0.630 n-butanol 0.425 0.004 0.0 0.490 methanol 0.090 0.139 0.0 0.594 methyl tert-butyl ether 0.483 0.105 0.142 0.000 hexane 1.000 0.000 0.000 0.000 heptane 1.152 0.000 0.000 0.000 water 0.000 0.000 0.000 1.000 Table S1. The NRTL-SAC parameters of the used solvents in this work. Journal of Chromatography A Volume 1393, 8 May 2015, Pages 47–56 Systematic and practical solvent system selection strategy based on the nonrandom two-liquid segment activity coefficient model for real-life counter-current chromatography separation Da-Bing Ren, Lun-Zhao Yi, Yan-Hua Qin, Yong-Huan Yun, Bai-Chuan Deng, Hong-Mei Lu, Xiao- Qing Chen, Yi-Zeng Liang, Solute X Y- Y+ Z Nicotinic acid 0.093 0.000 0.272 0.588 3,4-dihydroxybenzoic acid 0.338 0.728 0.708 0.742 Chlorogenic acid 0.142 1.074 1.086 1.416 Antipyrine 0.112 0.220 0.446 0.594 (Linear combination of) Eluotropic sequence parameters Polarity
  27. 27. Polarity 2. Solvatochromic (Reichardt’s) Dye
  28. 28. Hexanes Toluene t-BME Limonene Dichloromethane Dichloromethane Ethyl Acetate Acetone n-Butaniol Isopropanol Isopropanol Acetonitrile Ethanol Methanol Water
  29. 29. Solvatochromic (Reichardt’s) Dye https://upload.wikimedia.org/wikipedia/commons/thumb/2/28/Reichardt_dye_structure.svg/220px-Reichardt_dye_structure.svg.png
  30. 30. Solvatochromic (Reichardt’s) Dye 400 500 600 wavelengthinnm Upper Phase Lower Phase Reichardt’s dye lambda max values in the upper and lower phases of HEMWat solvent systems. Some values could not be determined due to insufficient solubility of the dye. Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085
  31. 31. 500 600 wavelength in nm Ch(Di)Mwat Solvent System DiMWat ChMWat Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085 Fig. 5. Reichardt’s dye lambda max values in the lower phases of ChMWat and DiMWat solvent systems. Solvatochromic (Reichardt’s) Dye
  32. 32. Polarity 3. GUESSmix
  33. 33. Model Compounds: HO H H H H O O OH OH O O O HO H H H CH3 OH OH O OHO OH N O OH O OH O HO O O OH O OHO O H HO H HO H H OHH O OH OH O OH O O OH OH OH O O OH OH HO O O H HO H HO H H OHH O OH OH N N N N O O N H O OH NH2 N N OH S O O O SO O O S O O O 3Na N H N O OH H H O O O O O O O O OH OH OH OOH HO The GUESSmix Friesen J.B, Pauli G.F. Journal of Liquid Chromatography and Related Technologies, 28: 2777-2806, 2005 b O Q r R U F Y C I E MZ V G T X H D N A
  34. 34. Solvent Systems Surveyed hexane ethyl acetate methanol chloroform dichloromethane water methyl t-butyl ether ACN Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085
  35. 35. GUESSmix polarity index (GUPI) 0 10 20 HepM 10:10 HterAcWat 8:2:8:2 HBuMWat 5:5:5:5 HEMWat 7:3:6:4 HEMWat 8:2:8:2 HEMWat 5:5:5:5 terBuMWat 5:5:5:5 HEMWat 4:6:4:6 DiMWat* 10:7:3 DiMWat* 10:6:4 terAcWat 7:3:10 terAcWat 7:6:7 HterAcWat 3:7:3:7 HterAcWat 6:10:1:4 HEMWat 1:9:1:9 HEMWat 3:7:3:7 ChWat* 10:10 ChMWat* 10:6:4 ChMWat* 10:4:6 ChMWat* 10:2:8 DiWat* 10:10 DiMWat* 10:4:6 DiMWat* 10:2:8 terAcWat 5:5:5 terAcWat 5:5:10 EBuWat 8:2:10 HterAcWat 5:5:5:5 EBuWat 4:6:10 terAcWat 8:2:10 number of GUESSmix compounds Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085 Fig. 1. Graphic representation of the GUESSmix polarity index (GUPI) of the solvent systems analyzed in this study. GUPI is the number of GUESSmix compounds with K values greater than 1 divided by the number of GUESSmix compounds with K values less than or equal to 1 in a reversed-phase elution method (aqueous or polar phase mobile). *The ChMWat and DiMWat solvent systems are represented as the inverse of the original K values.
  36. 36. Polarity 4. Phase Metering Apparatus
  37. 37. Phase Metering Apparatus 280000 280500 281000 281500 282000 282500 0 0.5 1 1.5 2 Log(Delectric Constant [literature values]) Pauli GF, Pro S, Chadwick L, Burdick T, Pro L, Friedl W, Novak, N, Maltby J, Qiu, F, Friesen JB Real-Time Volumetric Phase Monitoring Advances Chemical Analysis by Countercurrent Separation Analytical Chemistry 87:7418-7425 (2015) dx.doi.org/10.1021/acs.analchem.5b01613
  38. 38. Phase Metering Apparatus 300,500 302,500 304,500 2 PMA values time in min 301,500 302,500 303,500 304,500 2 6 PMA values time in min HEMWat 5:5:5:5 UP HEMWat 5:5:5:5 UP/LP 1:1 HEMWat 5:5:5:5 LP HEMWat 5:5:5:5 LP HEMWat 5:5:5:5 UP Pauli GF, Pro S, Chadwick L, Burdick T, Pro L, Friedl W, Novak, N, Maltby J, Qiu, F, Friesen JB Real-Time Volumetric Phase Monitoring Advances Chemical Analysis by Countercurrent Separation Analytical Chemistry 87:7418-7425 (2015) dx.doi.org/10.1021/acs.analchem.5b01613
  39. 39. Phase Metering Apparatus 300000 302000 304000 306000 upper lower Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085
  40. 40. Phase Metering Apparatus 300950 301950 302950 303950 304950 ChMWat DiMWat Upper Lower Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085
  41. 41. Phase Metering Apparatus 301900 302900 303900 HEMWat 10:0:10:0 HEMWat 9:1:9:1 HEMWat 8:2:8:2 HEMWat 7:3:7:3 HEMWat 7:3:6:4 HEMWat 6:4:6:4 HEMWat 7:3:5:5 HEMWat 6:4:5:5 HEMWat 5:5:5:5 HEMWat 4:6:5:5 HEMWat 3:7:5:5 HEMWat 4:6:4:6 HEMWat 3:7:4:6 HEMWat 3:7:3:7 HEMWat 2:8:2:8 HEMWat 1:9:1:9 HEMWat 0:10:0:10 ChMWat 10:0:10 ChMWat 10:1:9 ChMWat 10:2:8 ChMWat 10:3:7 ChMWat 10:4:6 ChMWat 10:5:5 ChMWat 10:6:4 ChMWat 10:7:3 DiMWat 10:0:10 DiMWat 10:1:9 DiMWat 10:2:8 DiMWat 10:3:7 DiMWat 10:4:6 DiMWat 10:5:5 DiMWat 10:6:4 DiMWat 10:7:3 EBuWat 10:0:10 EBuWat 8:2:10 EBuWat 6:4:10 EBuWat 4:6:10 EBuWat 2:8:10 EBuWat 0:10:10 Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085 Fig. 3. Averages of the upper and lower phase permittivity values measured by the phase metering apparatus for EBuWAt, DiMWat, ChMWat, and HEMWat.
  42. 42. Selectivity http://www.chocolati.com/new/images/P/24pcmix.jpg Solvent System Properties
  43. 43. Selectivity 60 160 PW PW+SS+PWSS Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085 Fig. 6. Selectivity parameters calculated for HEMWat solvent systems. Pairwise analysis calculated the ratio of K values for every pairwise combination. A ratio of greater than 5/3 or less than 3/5 is considered to be adequate for the resolution of two analytes. The number of resolved pairs is represented by αip. The number of GUESSmix analytes in the sweet spot is represented by Nsw. The number of resolved pairs in the sweet spot is represented by αsw. The sum of these three parameters (αip + Nsw + αsw), produced a selectivity score .
  44. 44. Selectivity 140 190 PW PW+SS+PWSS Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085 Fig. 6. Selectivity parameters calculated for DiMWat solvent systems. Pairwise analysis calculated the ratio of K values for every pairwise combination. A ratio of greater than 5/3 or less than 3/5 is considered to be adequate for the resolution of two analytes. The number of resolved pairs is represented by αip. The number of GUESSmix analytes in the sweet spot is represented by Nsw. The number of resolved pairs in the sweet spot is represented by αsw. The sum of these three parameters (αip + Nsw + αsw), produced a selectivity score .
  45. 45. Selectivity Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085 Fig. 6. Selectivity parameters calculated for terAcWat solvent systems. Pairwise analysis calculated the ratio of K values for every pairwise combination. A ratio of greater than 5/3 or less than 3/5 is considered to be adequate for the resolution of two analytes. The number of resolved pairs is represented by αip. The number of GUESSmix analytes in the sweet spot is represented by Nsw. The number of resolved pairs in the sweet spot is represented by αsw. The sum of these three parameters (αip + Nsw + αsw), produced a selectivity score .
  46. 46. Polarity and Selectivity 2D Reciprocal (Shifted) Symmetry Plots Mismatched Polarity Similar Polarity and Selectivity 2.7 4 8 8 2.7 4 8 8 Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085
  47. 47. Polarity and Selectivity 2D Reciprocal (Shifted) Symmetry Plots Similar Polarity with Different Selectivity Development and identification of orthogonal solvent systems is the key to the use of successive Countercurrent Separation steps in purification. 2.7 4 8 8 Friesen JB, Ahmed S, Pauli GF Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation Journal of Chromatography A 1377: 55-63 (2015) dx.doi.org/10.1016/j.chroma.2014.11.085
  48. 48. 1 0 0.5 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 2.5 3 3.5 4 HBuMWat 5:5:5:5 HterAcWat5:5:5:5 2.7 4 8 8 2.7 4 8 8 ChMWat 10:7:3 DiMWat10:7:3 0 0.5 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 2.5 3 3.5 42.7 4 8 8 2.7 4 8 8 0 0.5 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 2.5 3 3.5 4 DiMWat10:6:4 HEMWat 4:6:4:6 2.7 4 8 8 2.7 4 8 8 R C V/A M E Z UN F Q D DiMwat10:6:4 HterAcwat 4:6:4:6 0 0.5 1 1.5 2 2.5 3 3.5 4 0 0.5 1 1.5 2 2.5 3 3.5 42.7 4 8 8 2.7 4 8 8 R C M E Z U Q AV N F D A B C D
  49. 49. GUESSmix Polarity Matching
  50. 50. KD interval s 0 ≤ KD < 0.0625 0.062 5 ≤ KD < 0.125 0.125 ≤ KD < 0.25 0.25 ≤ KD < 0.5 0.5 ≤ KD < 1 1 ≤ KD < 2 2 ≤ KD < 4 4 ≤ KD < 8 8 ≤ KD < 16 16 ≤ KD < 32 32 ≤ KD < ∞ -8 rXHTDC GRFQUA VNMEZ OI Y b -7 rXHTDC GRFQUA VNE MZ O I Y b -6 rXHTDC GRFQUA VNE MZ O I Yb -5 rXHTD CGRAV FQU NE MZ O I Yb -4 rXHTD CGR AV FQ U NE M Z O I Yb -3 rXHTD CGR FQ U AVN E MZ O I Yb -2 rXHTD CGR FQ U AVN ME Z O I Yb -1 rXHTD CG RFQU AVN MEZ O I Yb 0 rXHTG DR CF QUA V N ME Z O I Yb
  51. 51. KD intervals 0 ≤ KD < 0.0625 0.0625 ≤ KD < 0.125 0.125 ≤ KD < 0.25 0.25 ≤ KD < 0.5 0.5 ≤ KD < 1 1 ≤ KD < 2 2 ≤ KD < 4 4 ≤ KD < 8 8 ≤ KD < 16 16 ≤ KD < 32 32 ≤ KD < ∞ 0 rXH TG DR CF QUA V N ME Z O I Yb +1 rXH T GR DC F QUA V NM EZ O I Yb +2 r XHT DCG RF QUA V NM EZ O I Yb +3 r XHT DCG RF QUA V M NE Z O IYb +4 rXH T DCG RFU QA V NM EZ O IYb +5 r XHT DC G RF UA V M QZ NEOIY b +6 r XHT D CG F RU AV M Z QNEO IYb +7 r XHT D CG F RUA V MZ QNEO IYb +8 rXH T D CG FRUA VM Z QNEO IYb Friesen JB, Pauli GF. Performance characteristics of countercurrent separation in analysis of natural products of agricultural significance. J. Agric. Food Chem., 56, 19-28, 2008
  52. 52. EBuWat Solvent System Family Ethyl Acetate / n-Butanol / Water 0 20 40 60 80 100 +5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5 solvent system number % Ethyl Acetate Butanol Water
  53. 53. Solvent System Maps Friesen, J.B. Pauli, G.F. Journal of Chromatography A 1151: 51-59 (2007) Sweet Spot FVAUMNE RZQOIYb GCDrTXH10:0:10 FVAUMNE RZQOIYb GDCXHTr8:2:10 FVAUMNE RZQOIYb GDCTXHr6:4:10 FVAUMNE RZQOIYb GCDTXHr4:6:10 FVAUMNE RZQOIYb GCDTXHr2:8:10 VAUMNER ZQOIYb FGCDTXHr0:10:10 32 ≤ KD < ∞ 16 ≤ KD < 32 8 ≤ KD < 16 4 ≤ KD < 8 2 ≤ KD < 4 1 ≤ KD < 2 0.5 ≤ KD < 1 0.25 ≤ KD < 0.5 0.125 ≤ KD < 0.25 0.062 5 ≤ KD < 0.125 0 ≤ KD < 0.0625 KD intervals Sweet Spot EBuWat
  54. 54. KD intervals 0 ≤ KD < 0.0625 0.063 ≤ KD < 0.125 0.125 ≤ KD < 0.25 0.25 ≤ KD < 0.5 0.5 ≤ KD < 1 1 ≤ KD < 2 2 ≤ KD < 4 4 ≤ KD < 8 8 ≤ KD < 16 16 ≤ KD < 32 32 ≤ KD < ∞ HEMW at 0 rXHTG DR CF QUAV N ME Z O I Yb DEMW at 0 rXHT G D C FUV A RQ ZMNE OI Yb DEMWat 0 (5:5:5:5) HEMWat 0 (5:5:5:5) FDR A O Yb Z E M NA VU X H T G r C Q I 0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1) ∞ A 280nm 230nm X H T r G D C F U V A R Q ZMNE OI Yb 0 0.25 0.5 0.75 1 1.33 2 4K'(1) SSMap ReSPlotReSPlot Pauli, G.; Pro, S.; Friesen, J. B. Countercurrent Separation of Natural Products. J. Nat. Prod. 2008, 71, 1489-1508
  55. 55. terAcWat system # relative proportions of solvents phase ratiot-butyl methyl ether acetonitrile water -5 10 0 10 45/55 -4 9 1 10 43/57 -3 8 2 10 41/59 -2 7 3 10 38/62 -1 6 4 10 36/64 0 5 5 10 34/66 +1 4 6 10 33/67 Solvent System Maps
  56. 56. QNEOI Yb MZAUFVRGCDXHTr 4:6:10 ZQNE OIYb UFMVARGCDrTXH 5:5:10 ZQNE OIYb UFMVARCDGrTXH 6:4:10 ZQNE OIYb UFMVARGCDrTXH 7:3:10 ZQNE OIYb UFMVARDGCrTXH 8:2:10 ZQNE OIYb UFMRVACDGrTXH 10:0:10 32 K < 16 K < 32 8 ? KD < 16 4 K < 8 2 ? KD < 4 1 ? KD < 2 0.5 ? KD < 1 0.25 ? KD < 0.5 0.125 K < 0.25 0.0625 K < 0.125 0 KD < 0.0625 K bins QNEOI Yb MZAUFVRGCDXHTr 4:6:10 ZQNE OIYb UFMVARGCDrTXH 5:5:10 ZQNE OIYb UFMVARCDGrTXH 6:4:10 ZQNE OIYb UFMVARGCDrTXH 7:3:10 ZQNE OIYb UFMVARDG ZQNE OIYb UFMRVA 32 Ki < ¥ 16 Ki < 32 8 Ki < 16 4 Ki < 8 2 Ki < 4 1 Ki < 2 0.5 Ki < 1 0.25 Ki < 0.5 0.125 Ki < 0.25 0.0625 £ Ki 0.125 0 £ Ki 0.0625 i Sweet Spot terAcWat SolventSystemMap of GUESSmixstandards £ £ £ £ £ £ £ £ £ G A V C C R X DN COOHOH HOOC OH OH O O OH OH N N N N O O CH3 CH3 H3C OHO HO OH HO O OH N H N O H3CO OCH3 O H3CO OCH3 OCH3 OCH3 OH OH O O O CH3 O OHO OCH3 OH OH N COOHOH HOOC OH OH O O OH OH N N N N O O CH3 CH3 H3C OHO HO OH HO O OH N H N O H3CO OCH3 O H3CO OCH3 OCH3 OCH3 OH OH O O O CH3 O OHO OCH3 OH OH
  57. 57. HterAcWat system # relative proportions of solvents phase ratiohexane tBME acetonitrile water -8 10 0 10 0 40/60 -7 9 1 9 1 41/59 -6 8 2 8 2 41/59 -5 7 3 7 3 43/43/14 -4 7 3 6 4 45/24/31 -3 6 4 6 4 44/28/28 -2 7 3 5 5 47/10/43 -1 6 4 5 5 46/14/40 0 5 5 5 5 51/11/38 +1 4 6 5 5 63/37 +2 3 7 5 5 66/34 +3 4 6 4 6 58/42 +4 3 7 4 6 59/41 +5 3 7 3 7 55/45 +6 2 8 2 8 50/50 +7 1 9 1 9 49/51 +8 0 10 0 10 45/55
  58. 58. KD intervals 0 ≤ KD < 0.0625 0.0625 ≤ KD < 0.125 0.125 ≤ KD < 0.25 0.25 ≤ KD < 0.5 0.5 ≤ KD < 1 1 ≤ KD < 2 2 ≤ KD < 4 4 ≤ KD < 8 8 ≤ KD < 16 16 ≤ KD < 32 32 ≤ KD < ∞ 10:0:10:0 rTXHGDC RFVUAQN EMZ O I Y b 8:2:8:2 rTXHGDC RFVUAQN E MZ O I Yb 4:6:5:5 rTXH DCG RF AVU Q M ZN E OIYb 4:6:4:6 rTXH DCG RF AVU Q M ZN E OIYb 3:7:3:7 rTXH DC G RF AV U M ZQ NE OIYb 2:8:2:8 rTXH DC G RA FVU M ZQNE OIYb 0:10:0:10 rTXH DCG RAV FUM ZQNE OIYb Sweet Spot HterAcWatSolvent System Map
  59. 59. Comparing SS in different families r T X H C D G R V A F U M Z Q N E O I Y b c) terAcWat6:4:10 polar r T X H D C G V A F U M N E R Z Q O I Y b nonpolar a) EBuWat r T X H C D G R F V A U Q M Z N E O I Y b d) HterAcWat4:6:4:6 r T X H C D G R V A F U M Z Q N E O I Y b b) terAcWat Friesen JB, Pauli GF. Rational development of solvent system families in counter-current chromatography. J. Chromatogr. A, 1151, 51-59, 2007;

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