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Solvent System Selection

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Description of the different approaches leading to the selection of bi(tri)phasic solvent systems to be utilized in countercurrent chromatography.

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Solvent System Selection

  1. 1. Solvent System Selection The 9th International CCC Event in Chicago/USA Conference: August 1-3, Workshop: July 30-31, 2016 Dominican University, River Forest, IL (U.S.A.) J. Brent Friesen, Chemistry Professor, Dominican University jbfriesen@dom.edu
  2. 2. TI S n a t u r a l a a l n r t u Solvent Systems (SSs) Instrumentation Theory Empirical Approach Rational Approach Historic SSs Workhorse SSs Modified SSs Structure & Polarity G.U.E.S.S. SS Mapping Ito Machines Current Market Craig Machines Operation Modes Complete Recovery Priority of K Calculation & Modeling KD VCM VC VM VS Sf b F VR f G Guidelines S-I-T selection sym ReSS Plot 0 ¥ R Reporting S-I-T documentation Pauli, G.; Pro, S.; Friesen, J. B. Countercurrent Separation of Natural Products. J. Nat. Prod. 2008, 71, 1489-1508
  3. 3. Selectivity left fieldcenter fieldright field HPLC
  4. 4. good 0.25 < K < 16 spot sweet not good K < 0.25 not good 16 < K Absorbance volume sweet spot CCS Selectivity
  5. 5. Liu Y, Friesen JB, McAlpine JB, Pauli GF Solvent System Selection Strategies in Countercurrent Separation Planta Medica 81: 1582-1591 (2015) dx.doi.org/10.1055/s-0035-1546246
  6. 6. Survey Results 222 50 16 7 3 1 2 3 4 5-7 Number of SS per publication numberofSSs Friesen 2015 J Nat Prod vol. 78 p.1764
  7. 7. Survey Results 94 44 37 18 13 12 1 1 2 3 4 5 6-10 11-30 SS Families Used in SS Selection numberofSSfamilies number of articles 6 14 42 39 27 21 12 15 11 30 1 2 3 4 5 6 7 8 9 10 11-30 31-63 SS Used in SS Selection numberofSS number of articles Friesen 2015 J Nat Prod vol. 78 p.1764
  8. 8. Where to start? Where do solvent systems come from? http://riverbankoftruth.com/wp-content/uploads/2013/04/76149_231332106997173_1550272648_n.jpg
  9. 9. SeparationTables Counter-current chromatography for the separation of terpenoids: a comprehensive review with respect to the solvent systems employed Krystyna Skalicka-Woz´niak • Ian Garrard DOI 10.1007/s11101-014-9348-2 Phytochem Rev (2014) 13:547–572 Where to start?
  10. 10. Where to start? The usual suspects: portal or scout solvent systems 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 ethanol : phosphate buffer 8 hexane : methyl t-butyl ether : acetonitrile 10:1:10 HterAc 9 dichloromethane ethyl acetate : methanol : water 5:5:5:5 DEMWat 10 hexane : methyl t-butyl ether : methanol : water 5:5:5:5 HterMWa
  11. 11. First Round Attempt(s) 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
  12. 12. Ginger Mass Partitioning* 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 8 1 3 10 2 6 5 4 9 7 GG lipo GG hydro * 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 1 2 3 4 5 6 7 8 9 10 Ginger Mass Partitioning GGU GGL Ginger1 2 3 4 56 78 9 10 * 1. HAcM 10:5:5 2. HterAcWat 8:2:8:2 3. HEMWat 5:5:5:5 4. ChMWat 10:7:3 5. terAcWat 4:6:10 6. EBuWat 4:6:10 7. 1-propanol:800 mM aqueous potassium phosphate buffer (pH 7.4) 8. HterAcWat 10:1:10 9. DEMWat 5:5:5:5 10. HterMWat 5:5:5:5
  13. 13. * HEMWat Ginger1 2 3 4 56 78 9 10
  14. 14. Small-scale CCS scouting Fig. 2. Parallel strategy for rapid screening of biphasic liquid systems for E. rutaecarpa extract (a) Arizona liquid system J; (b) Arizona liquid system N; (c) Arizona liquid system S; (d) Arizona liquid system P; (e) Arizona liquid system Q; and (f) Arizona liquid system R. Column capacity of 40mL for each. Flow rate: 2.0 mL/min of lower aqueous phase in the head-to-tail (descending) direction, up to VCM (20 mL) immediately followed by 2 mL/min of upper phase flowing in the same direction; revolution speed: 650 rpm; detection: 254 nm; injected sample: 10mg crude extract dissolved in 1mL upper phase and 1mL lower phase. Rapid screening of bioactive components from Zingiber cassumunar using elution-extrusion counter-current chromatographyJournal of Chromatography A, Volume 1181, Issues 1–2, 15 February 2008, Pages 33-44 Yanbin Lu, Rui Liu, Alain Berthod, Yuanjiang Pan
  15. 15. Solvent system screening of standard compounds sulfamethazine (1), reserpine (2), and flavone (3). All separations were performed in elution-extrusion mode using the lower phase of the solvent system as the mobile phase at 2 mL/min. Detection was by MS (APCI)–TIC. 19:1:19:1 3:2:3:2 2:5:2:5 J Sep Sci. 2015 Dec;38(23):3983-91. doi: 10.1002/jssc.201500574. Automated solvent system screening for the preparative countercurrent chromatography of pharmaceutical discovery compounds. Bradow J, Riley F, Philippe L, Yan Q1, Schuff B, Harris GH. Small-scale CCS scouting
  16. 16. Systematic search for suitable solvent systems for HSCCC. A set of solvent systems is arranged from the top to the bottom according to a decreasing order of the hydrophobicity of their organic phases. When the hydrophobicity of the target analyte is unknown, the search may start at hexane–ethyl acetate–methanol–water (3:5:3:5, v/v) and then follow the direction indicated by a pair of arrows. Importantly, each of these solvent systems gives nearly a 1:1 phase volume ratio, conserving the solvent in CCC and making it easier to separate the two phases both during evaluation in the test tube, and when preparing phases in a separatory funnel. 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 selection of this two-phase solvent system for the target compound(s) is the most important step in HSCCC where searching for a suitable two-phase solvent system may be estimated as 90% of the entire work in HSCCC.” Ito’s Golden Rules
  17. 17. Ito’s Golden Rules Rule 1: First study previous articles on the CCC or CCD Rule 2: Find systems with K values of the target compounds in a proper range: The suitable K values for HSCCC are 0.5≤K≤1.0. Rule 3: Search systematically for a suitable solvent system according to Tables 1 and 2 Rule 4: Modify the K value of the negatively charged analytes, e.g., carboxylic acids, by adding acids such as TFA and acetic acid to the solvent system. Rule 5: The ratio of the two K values or the separation factor (α = K1/K2, where K1 >K2) ought to be greater than 1.5. Rule 6: If K is 1, the analyte will elute at the retention volume equal to the column capacity regardless of the retention volume of the stationary phase. At K <1 or K> 1, the analyte would elute before or after the elution of one column volume, respectively. Rule 7: The higher the retention of the stationary phase, the better the peak resolution. Rule 8: Measure the settling time of the two- phase solvent system to be used for the separation. Rule 9: Ensure that the sample solution forms two phases when mixed with either phase in the column. Rule 10: A lower (heavier) mobile phase should be introduced through the head à tail, and an upper (lighter) mobile phase in the opposite direction. This is extremely important because the elution of either phase in the wrong direction results in an almost complete loss of the stationary phase from the column. Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito
  18. 18. Ito’s Golden Rules Rule 11: Avoid trapping the air in the column especially in a preparative column. Rule 12: Avoid trapping the stationary phase in the vertical flow cell by eluting the lower mobile phase upward from the bottom and the reverse if the upper is used as the mobile phase. http://taylormarshall.com/wp-content/uploads/2013/08/charlton-heston-10-Commandments-Moses-Horns.jpg Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito
  19. 19. A Generally Useful Estimation of Solvent Systems in CCS
  20. 20. 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
  21. 21. § Commercially available natural products Choosing Model Compounds § Absorb in UV-vis Umbelliferone Absorbance values in HEMWat +2 0 0.5 1 260 285 310 335 360 nm A upper phase lower phase P value § Revealed by a common TLC revelation methods § Inexpensive § Available in high purity § Relatively non-toxic § Varied polarity § Varied functional groups http://www.univ-lille1.fr/sajec2005/SigmaAldrich.gif
  22. 22. Trends in K Umbelliferone P values in HEMWat systems 0 10 20 30 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 HEMWat number Pvalue •Due to the exponential relationship, a logarithmic plot represents a linear correlation between the solvent system number and LogK. Umbelliferone LogP values in HEMWat systems y = 0.23x - 0.4905 -2 -1 0 1 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 HEMWat number LogP •An increase in K is observed as the HEMWat solvent system becomes more positive. Friesen, J. B.; Pauli, G. F., GUESS - A generally useful estimate of solvent systems in CCC. Journal of Liquid Chromatography and Related Technologies 2005, 28, 2777-2806.
  23. 23. -2 -1.2 -0.4 0.4 1.2 2 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 LogK HEMWat number Partition Coefficients in HEMWat: LogK Cholesterol Stigmasterol Ionone Carvone Reserpine Salicylic Coumarin Estradiol Naringenin Vanillin Aspirin Umbelliferone Quercetin Ferulic Acid Caffeine Tryptophan Nicotinic Acid Tannic Acid Chlorogenic Acid G.U.E.S.S. Standards The ideal HEMWat number is the HEMWat solvent system that gives LogK = 0. Friesen, J. B.; Pauli, G. F., GUESS - A generally useful estimate of solvent systems in CCC. Journal of Liquid Chromatography and Related Technologies 2005, 28, 2777- 2806.
  24. 24. Predicting K from HEMWat one HEMWat SS to another Average slope of LogK plot is 0.16 Slope = 0.16 = (y2-y1)/(x2 – x1) LogKb = LogKa + 0.23(b-a) If you know Ka in SS “a” what is Kb in SS “b” ?
  25. 25. Predicting K from HEMWat one HEMWat SS to another Average slope of LogK plot is 0.18 Slope = 0.18 = (y2-y1)/(x2 – x1) = (LobKb-LogKa)/(b-a) LogK-3 = 0.038 If you know Ka in HEMWat #a what is Kb in in HEMWat #b ? LogKb = LogKa + 0.18(b-a) Carvone has a K value of 4.0 in HEMWat 0. What is its K value in HEMWat -3? K-3 = 1.09 (actual value 1.57) LogK-3 = LogK0 + 0.18(-3-0) = Log(4.0) + 1.8(-3-0) = 0.602-0.54
  26. 26. TLC: the eyes of the Organic Chemist
  27. 27. Use the organic phase of biphasic solvent system as TLC developing solvent Hostettmann, K.H.-K.M.; Sticher, O. Application of droplet CCC to the isolation of natural products. J. Chromatogr. A 1979, 186, 529–534.
  28. 28. G.U.E.S.S. Overview Generally Useful Estimation of Solvent Systems combines the convenience of TLC with the separation power of HSCCC. Friesen, J. B.; Pauli, G. F., GUESS - A generally useful estimate of solvent systems in CCC. Journal of Liquid Chromatography and Related Technologies 2005, 28, 2777-2806. H E M Wat G.U.E.S.S. HSCCC
  29. 29. G.U.E.S.S. theory Absorbance volume K = 1 K = 0.25 K = 4 Rf = 0.5 Rf = 0.6 Rf = 0.4 sweet spot
  30. 30. TLC “SSE” Solvent Systems HEMWat nHex EtOAc MeOH Water SSE nHex EtOAc –7 9 1 9 1 1 9 1 –6 8 2 8 2 2 8 2 -5 7 3 7 3 3 7 3 -4 7 3 6 4 3 7 3 -3 6 4 6 4 4 6 4 -2 7 3 5 5 3 7 3 -1 6 4 5 5 4 6 4 0 5 5 5 5 5 5 5 +1 4 6 5 5 6 4 6 +2 3 7 5 5 7 3 7 +3 4 6 4 6 6 4 6 +4 3 7 4 6 7 3 7 +5 3 7 3 7 7 3 7 +6 2 8 2 8 8 2 8 +7 1 9 1 9 9 1 9 +8 0 10 0 10 10 0 10 In order to simplify the TLC, The HEMWat organic phase can be replaced with a solvent system similar to the HEMWat organic phase that can be mixed without the tedious equilibrating of a biphasic system. There are 16 HEMWat systems that correspond to only 10 SSE (hexane/ethyl acetate) systems. Friesen, J. B.; Pauli, G. F., GUESS - A generally useful estimate of solvent systems in CCC. Journal of Liquid Chromatography and Related Technologies 2005, 28, 2777-2806.
  31. 31. 10:0:10:0 9:1:9:1 8:2:8:2 7:3:7:3 6:4:6:4 5:5:5:5 4:6:4:6 3:7:3:7 2:8:2:8 1:9:1:9 0:10:0:10 7:3:6:4 7:3:5:5 6:4:5:5 3:7:5:5 4:6:5:5 3:7:4:6 HEMWat family white and black keys 10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10 HEHEMWat
  32. 32. G.U.E.S.S. considerations 1. What is the range ? < Rf < ? 2. What is the range ? < K < ? 3. How good is Rf à K correlation? 4. Is organic phase TLC elution a better approximation than the SS scale?
  33. 33. GUESS Applications CCC conditions TLC conditions TLC Rf values ChMWat CHCl3:CH3OH:H2O SSC CHCl3:CH3OH:H2O 1 2 3 +1 100:40:60 5 90:10:0.5 0.16 0.16 0.20 +2 100:50:50 6 85:15:0.5 0.48 0.48 0.54 +3 100:60:40 7 80:19:1.0 0.63 0.65 0.69 1 N OH OH O N OH O HO N O OH HO 1 2 3 5' 4' 3 6 7 8 9 10 11 12 13 14 15 16 Fraction Normalized Abundance 3 2 1 Liu, Y.; Friesen, J. B.; Klein, L. L.; McAlpine, J. B.; Lankin, D. C.; Pauli, G. F.; Chen, S.-N., The Generally Useful Estimate of Solvent Systems (GUESS) method enables the rapid purification of methylpyridoxine regioisomers by countercurrent separation. Journal of Chromatography A 2015, 1426, 248-251.
  34. 34. other solvent systems? LogP in ChMWat -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 -3 -2 -1 0 1 2 3 4 ChMWat number LogP Coumarin Estradiol Vanillin Quercetin Caffeine Nicotinic Acid Chorogenic Tannic Acid Salicin ChMWat CHCl3 CH3OH H2O SSC CHCl3 CH3OH H2O -3 100 0 0 1 100 0 0 -2 100 10 90 2 99 1 0 -1 100 20 80 3 98 2 0 0 100 30 70 4 95 5 0 +1 100 40 60 5 90 10 0.5 +2 100 50 50 6 85 15 0.5 +3 100 60 40 7 80 19 1 +4 100 70 30 8 75 24 1 Friesen, J. B.; Pauli, G. F., GUESS - A generally useful estimate of solvent systems in CCC. Journal of Liquid Chromatography and Related Technologies 2005, 28, 2777-2806.

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