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Optimization parameters in Countercurrent Chromatography

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The different parameters to modify or control in order to optimize the separation of target compounds in countercurrent chromatography.

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Optimization parameters in Countercurrent Chromatography

  1. 1. Optimization https://photos.prnewswire.com/prnvar/20160223/336432
  2. 2. Sample Preparation Equal amounts of upper and lower phases. (Usually best for overall solubility) Add to sample loop in alternating plugs Leave a buffer of mobile phase on each side of the sample loop Need to filter insoluble material?
  3. 3. Column Loading http://www.microcontractor.org/uploads/4/4/9/7/44977385/8245406_orig.jpg 1. Column loading - higher sample concentration - more sample volume 2. scale-up
  4. 4. Preparative loading of CCS instruments 322 articles 52 journals 2008 through 2012. 15 52 27 24 4 0 10 20 30 40 50 60 < 100 101-500 501-1000 1001-5000 > 5000 number of separations mg sample 52 133 53 49 17 0 20 40 60 80 100 120 140 < 100 101-500 501-1000 1001-5000 > 5000 number of separations mg sample Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796. Pauli, G. F.; Pro, S. M.; Friesen, J. B., Countercurrent separation of natural products. Journal of Natural Products 2008, 71, 1489-1508. 2000 through 2007 2008 through 2012 137 articles 24 journals 2000 through 2007. Column Loading
  5. 5. Column Loading 15 40 61 102 15 9 0.01 - 0.20 0.21 - 0.50 0.51 - 1.00 1.01 - 5.0 5.2 - 10.0 10.5 - 21.0 number of separations mg sample /mL column volume Column Loading: HSCCC instruments 0.01 - 0.20 0.21 -0.50 0.51 - 1.00 1.01 - 5.0 5.2 - 10.0 10.5 - 375 B 21 26 129 66 44 18 Column Loading: All CS instruments number of separations Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  6. 6. Column Loading 0 2000 4000 6000 8000 10000 12000 14000 16000 0 50000 100000 150000 200000 250000 v o l u m e m L mg loaded mg loaded & volume 0 200 400 600 800 1000 1200 1400 1600 1800 0 2000 4000 6000 8000 v o l u m e m L mg loaded mg loaded & volume Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  7. 7. 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
  8. 8. GUESSmix Used to Evaluate SSs The distribution coefficient (K) is a constant for a particular substance in a particular solvent system. Independent of: - column volume - instrument model - normal or reverse-phase mode - run time - rotation speed - flow rate G Mix H/tBME/ACN/Water 4:6:5:5 10/12/06 -0.1 0.8 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 mL & mn A 280nm 230nm K-Based Chromatography
  9. 9. CCC Chromatogram 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 A254 mL GUESSmix in ChMWat 10:7:3 12/14/12, 35 degrees N Phase Compounds were identified using TLC and CCC peaks Q HD FUE C V r
  10. 10. HPLC • Next, HPLC was used to further identify compounds along with the TLC plates and GC chromatograms -56.05 43.95 143.95 243.95 343.95 443.95 543.95 643.95 743.95 843.95 943.95 1043.95 1143.95 1243.95 1343.95 1443.95 0 5 10 15 20 25 30 35 40 45 50 55 60 65 X T Gr 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 A254 tube GUESSmixin DMWat10:6:4 11/02/12, 35degreesN Phase G T r X HPLC of test tube 69 69 69
  11. 11. Emphasize the importance of K Using GUESSmix to explore solvent system families. Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007) Symmetry Midline 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K A M Q V U F N Z E
  12. 12. Column Loading What is the effect of column loading on K value and resolution?
  13. 13. 10mg/compound 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 5 mg/compound 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 20mg/compound 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Sf = 0.52 Sf = 0.54 Sf = 0.56 Column Loading
  14. 14. 30mg/compound 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 50mg/compound 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 40mg/compund 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Sf = 0.59 Sf = 0.59 Sf = 0.44 Column Loading
  15. 15. Column Loading Solute-solute interactions may decrease or increase resolution. Solute-solute interactions may affect the apparent (experimental) K value.
  16. 16. Fig. 3. APCI-MS (pos. mode) molecular weight profile chromatogram generated by sequential injections of even numbered fractions of the HPCCC preparative separations of S. terebinthifolius berries on the Midi and selected ion traces of peaks 1–3. Each signal contained the complete APCI-MS profile information of compounds present in a single assay tube (distance between two monitoring signals is equivalent to 50.0 mL – Midi I to III and 80 mL – Midi IV). J Chromatogr A. 2015 Apr 10;1389:39-48. doi: 10.1016/j.chroma.2015.02.005. Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High performance countercurrent chromatography fractionation of triterpene acids with off- line detection using atmospheric pressure chemical ionization mass spectrometry. Vieira MN, Costa Fd, Leitão GG, Garrard I, Hewitson P, Ignatova S, Winterhalter P, Jerz G Column Loading n-heptane/ethyl acetate/methanol/water (6:1:6:1)
  17. 17. Figure 4. HSCCC chromatogram of crude flavonol glycosides from Ginkgo biloba leaves at flow rate 1.2 mL/min. n-hexane/butanol/ethyl acetate/methanol/0.5% acetic acid (2:1:7:2:8) J. Sep. Sci. 2007, 30, 2153 – 2159 Qiang Zhang, Li-Juan Chen, Hao-Yu Ye, Lei Gao, Wenli Hou, Minghai Tang, Guangli Yang, Zhenhua Zhong, Yuan Yuan, Aihua Peng, Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography Column Loading
  18. 18. (a) HSCCC chromatogram when injection volume was 100 mg for MD-R. (b) HSCCC chromatogram when injection volume was 600 mg for MD-R. HEMWat 10:2:5:7, 800 rpm, lower phase mobile 2 mL/mi. 280 nm TBE300 J Chromatogr B 2016 Feb 1;1011:99-107. doi: 10.1016/j.jchromb.2015.12.051. Separation and preparation of 6-gingerol from molecular distillation residue of Yunnan ginger rhizomes by high-speed counter-current chromatography and the antioxidant activity of ginger oils in vitro. Gan Z, Liang Z, Chen X, Wen X, Wang Y, Li M, Ni Y. Column Loading
  19. 19. Phytochem Anal. 2015 Nov-Dec;26(6):444-53. doi: 10.1002/pca.2579. Rapid Separation of Three Proanthocyanidin Dimers from Iris lactea Pall. var. Chinensis (Fisch.) Koidz by High-Speed Counter-Current Chromatography With Continuous Sample Load and Double-Pump Balancing Mode. Lv H, Yuan Z, Wang X, Wang Z, Suo Y, Wang H. Figure 5. HSCCC chromatograms of the EPS at different load masses. HSCCC conditions: solvent system: EBuWat (9:1:10); revolution speed: 900 rpm; separation temperature: 30 °C; flow rate: 2.2mL/min; detection wavelength: 280 nm; sample size: (A) 50mg, (B) 100mg, (C) 150mg, (D) 200mg of the EPS in 5mL of the upper phase and 5 mL of the lower phase. Column Loading
  20. 20. Scale-Up http://ian.umces.edu/imagelibrary/albums/userpics/12789/normal_ian-symbol-mountains-snowcaps-and-foothills.png
  21. 21. Fig. 6. Comparison of chromatograms for different coils operated in similar conditions with their corresponding parameters in the reversed-phase mode. (A) The chromatogram for the 4.7 ml coil on Mini-DE: HepEMWat phase system of 2:3:2:3; rotation speed, 2000 rpm; sample loop, 0.1ml; sample concentration, 20 mg/ml; flow rate, 1.0ml/min; temperature, 25 ◦C; Sf = 53.03%. (B) The chromatogram for the 17.2 ml coil on Mini-DE: HepEMWat phase system of 2:3:2:3; rotation speed, 2000 rpm; sample loop, 0.43 ml; sample concentration, 20 mg/ml; flow rate, 1.0ml/min; temperature, 25 ◦C: Sf = 53.01%. (C) The chromatogram for the 915.5 ml coil on Midi-DE: HepEMWat phase system of 2:3:2:3; rotation speed, 1250 rpm; sample loop, 20.0 ml; sample concentration, 20 mg/ml; flowrate, 50.0 ml/min; temperature, 25 ◦C; Sf = 53.33%. Journal of Chromatography A, 1194 (2008) 192–198 How to realize the linear scale-up process for rapid purification using high-performance counter-current chromatography Yuan Yuan, BiqinWang, Lijuan Chen, Houding Luo, Derek Fisher, Ian A. Sutherland,Yuquan Wei Scale-up
  22. 22. Scale-up of counter-current chromatography: Demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale Journal of Chromatography A, Volume 1216, Issue 50, 11 December 2009, Pages 8787-8792 Ian Sutherland, Peter Hewitson, Svetlana Ignatova Scale-up Fig. 1. 4.6 L Maxi separation of benzyl alcohol and p-cresol scaled up 850× from the Milli-CCC separation (inset). Operating conditions for 4.6 L Maxi: speed 600 rpm, flow 850 mL/min; sample load, 290mL (6.3% Vc) 12.2 g BA, 5.8 g PC. Run conditions for the Milli-CCC: speed 2100 rpm, flow 1 mL/min; sample loading condition the same concentration and proportion of column volume as for Maxi. Phase system: HEMWat (14:1:5:10, v/v/v/v). Sf before injection 80.0%, after separation 40.8% for the Maxi run.
  23. 23. Purity values and centrifugal partition chromatography elution profile of the major compounds isolated from a crude aqueous extract of Stevia leaves using the ASCPC250® instrument (experiment A, 500 mg injected; experiment B, 1 g injected) or the FCPE300® instrument (experiment C, 5 g injected). Sf = 0.75, 0.70 & 0.70 for A, B & C. 1200 rpm A & B, 1000 rpm C. 10 mL/min A & B, 20 mL/min C. Planta Med. 2015 Nov;81(17):1614-20. doi: 10.1055/s-0035-1545840. Intensified Separation of Steviol Glycosides from a Crude Aqueous Extract of Stevia rebaudiana Leaves Using Centrifugal Partition Chromatography. Hubert J, Borie N, Chollet S, Perret J, Barbet-Massin C, Berger M, Daydé J, Renault JH. Scale-up
  24. 24. Sf
  25. 25. Figure 3.2 The importance of the amount of stationary phase retained in a 100 ml CCC column (vertical dotted line). Chromatograms of the same mixture of seven solutes (listed in Table 3.1) obtained with the same CCC hydrodynamic column and the same biphasic liquid system: hexane/ethyl acetate/methanol/water 4:6:4:6 v/v. Aqueous lower mobile phase, 2 ml/ min, rotor rotation 900 rpm, average efficiency ∼300 plates Separations with a Liquid Stationary Phase: Countercurrent Chromatography or Centrifugal Partition Chromatography Alain Berthod and Karine Faure Analytical Separation Science, First Edition. Edited by Jared L. Anderson, Alain Berthod, Verónica Pino Estévez, and Apryll M. Stalcup. 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Sf
  26. 26. 6 5 16 42 68 73 44 26 2 0 10 20 30 40 50 60 70 80 10-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 reported Sf values Sf Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  27. 27. Determination of Sf • (i) the carry-over method Sf(CO): this approach measures, in a graduated cylinder, the amount of stationary phase carried over as the column is equilibrated with the mobile phase. The amount of stationary phase displaced is called the “carry over volume” or V(CO). In the case of large volume columns, V(CO) is considered to be equal to the mobile phase volume inside the column (VM). • (ii) void volume determination by UV detection Sf(UV): this method is routinely employed with crude natural extracts, is to identify the mobile phase front by unretained UV-active sample components that are almost always present in complex natural mixtures. The void volume (V(UV)) is determined by taking the analyte retention volume (VR) of an unretained component(s) (VR 0) to be equal to VM. • (iii) volumetrics of extruded mobile phase Sf(MP): the third method is based on the determination of VM by measuring the volume of mobile phase extruded (V(MP)) from the column after the separation is complete.
  28. 28. Flow Rate https://farm7.staticflickr.com/6081/6077573063_2bdf56e975_z.jpg
  29. 29. 600m rpm 2 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 600 rpm, 1.5 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 600 rpm, 3 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.52 Sf = 0.47 Sf = 0.36 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Increasing Flow Rate (constant rpm) Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  30. 30. Sf and Flow Rate Fig. 3. Stationary (upper) phase retention ratio in percentage of the column volume plotted versus the square root of the lower mobile phase flow rate. CCC column volumes and rotor rotations: Mini 1.6mm I.D. 20.8mL and 1800 rpm; Mini 0.8mm I.D. 19.5mL and 2100 rpm; SFCC 1-coil 54mL and 800 rpm; SFCC 3-coil 156mL and 800 rpm. HepEMWat 2:3:2:3, head to tail flowing direction. The regression equations give A intercepts and B slopes (Eq. (5)). The R2 regression coefficient is listed below its equation. Berthod2009_JCA_1216_4169_SmallVolume
  31. 31. CPC Fig. 4. Evolution of the stationary phase (Sf) retention as a function of flow rate (F) for different rotational speeds; Sf0, y-abscises intercept, corresponds to the cells fraction of the column volume. Dots are experimental data; straight lines show tendency and flooding transition J Chromatogr A. 2015 Apr 24;1391:80-7. doi: 10.1016/j.chroma.2015.03.005. Modeling pH-zone refining countercurrent chromatography: a dynamic approach. Kotland A, Chollet S, Autret JM, Diard C, Marchal L, Renault JH. Sf and Flow Rate
  32. 32. Flow Rate Fig. 3. (a–c) Separation of the GUESS mixture as described in [15] using DE Spectrum preparative coils at 80×g (918 rpm), 136mL column volume, HEMWat (2:3:2:3), lower layer as mobile phase, 1.5, 3 and 6mL/min, head to tail elution, Sf = 77 % (1.5mL/min), 64% (3 mL/min) and 53% (mL/min); 60mg total loading (5mg of each 12 standards: aspirin (A), -carotene, caffeine (C), nicotinic acid (D), estradiol, ferulic acid (F), naringenin, carvone, red new Coccine (R), quercetin (Q), umbelliferone (U), vanillin (V)), monitored by DAD 254 nm, compared separation times 130, 60 and 32 min, respectively. 1.5 mL/min Sf = 0.77 3 mL/min, Sf = 0.64 6 mL/min Sf = 0.64 918 rpm Performance comparison using the GUESS mixture to evaluate counter-current chromatography instruments Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4181-4186 Hacer Guzlek, Philip Leslie Wood, Lee Janaway
  33. 33. Flow Rate Fig. 4. Separation of the GUESS mixture using DE Spectrum preparative coils at 243×g (1600 rpm), 136mL column volume, HEMWat (2:3:2:3), lower layer as mobile phase, 1.5, 3 and 6 mL/min, head to tail elution, Sf = 88% (1.5 mL/min), 85% (3 mL/min) and 73% (6 mL/min); 60mg total loading for 1.5 and 6mL/min; total loading for 3 mL/min was 55mg because red new Coccine was not available (5mg of each 12 standards: aspirin (A), -carotene, Caffeine (C), nicotinic acid (D), estradiol, ferulic acid (F), naringenin, carvone, red new Coccine (R), quercetin (Q), umbelliferone (U), vanillin (V)), monitored by DAD 254 nm, compared separation times 105, 60 and 30 min, respectively. 1600 rpm 1.5 mL/min Sf = 0.88 3 mL/min, Sf = 0.85 6 mL/min Sf = 0.73 Performance comparison using the GUESS mixture to evaluate counter-current chromatography instruments Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4181-4186 Hacer Guzlek, Philip Leslie Wood, Lee Janaway
  34. 34. Figure 3. HSCCC chromatogram of crude flavonol glycosides from Ginkgo biloba leaves. HSCCC conditions: column volume: 40 mL; solvent system: hexane/butanol/ethyl acetate/methanol–0.5% acetic acid (1:0.5:3.5:1:4); stationary phase: lower; 1600 rpm; detection wavelength: 254 nm; temperature: 25 oC; sample concentration: 20 mg/mL; Sf phase at 1.0, 1.2, and 1.5 mL/min flow rates: 78.1, 60.1, and 45.4%, respectively. J. Sep. Sci. 2007, 30, 2153 – 2159 Qiang Zhang, Li-Juan Chen, Hao-Yu Ye, Lei Gao, Wenli Hou, Minghai Tang, Guangli Yang, Zhenhua Zhong, Yuan Yuan, Aihua Peng, Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography Flow Rate
  35. 35. Phytochem Anal. 2015 Nov-Dec;26(6):444-53. doi: 10.1002/pca.2579. Rapid Separation of Three Proanthocyanidin Dimers from Iris lactea Pall. var. Chinensis (Fisch.) Koidz by High-Speed Counter-Current Chromatography With Continuous Sample Load and Double-Pump Balancing Mode. Lv H, Yuan Z, Wang X, Wang Z, Suo Y, Wang H. Figure 4. HSCCC chromatograms of the EPS at different flow rates. HSCCC conditions: EBuWat (9:1:10): 900 rpm: 30 °C;: 50mg of the EPS in 5mL of the upper phase and 5mL of the lower phase; detection 280nm; flow rate: (A) 1.2mL/min, (B) 1.5mL/min, (C) 1.8mL/min, (D) 2.2mL/min. Flow Rate Gradients
  36. 36. Figure 3. HPCCC separation patterns of the acetone-soluble extract of fermented C. sinensis leaves using HEMWat (1:9:1:9, v/v) system with rotational speed at 1600 rpm. (A) flow-rate at 3.0 mL/min; (B) flow-rate at 5.0 mL/min; (C) flow-rate at 8.0 mL/min; (D) gradient flow-rate at 3.0 mL/min in 0–45 min, and 5.0 mL/min in 45– 130 min. Peaks 1: caffeine, 2: (−)-epigallocatechin 3-O-gallate, 3: (−)-gallocatechin 3- O-gallate, and 4: (−)-epicatechin 3-O-gallate. Molecules 2015, 20, 13216-13225; doi:10.3390/molecules200713216 Separation of Polyphenols and Caffeine from the Acetone Extract of Fermented Tea Leaves (Camellia sinensis) Using High-Performance Countercurrent Chromatography Soo Jung Choi, Yong Deog Hong, Bumjin Lee, Jun Seong Park 2, Hyun Woo Jeong, Wan Gi Kim, Song Seok Shin and Kee Dong Yoon Flow Rate Gradients
  37. 37. Fig. 3. HSCCC chromatograms of the EFS. Peak 1: procyanidin B3, Peak 2: procyanidin B1, Peak 3: catechin, and Peak 4: procyanidin B7. HSCCC conditions: solvent system: HEMWat (0.75:12.5:1:12.5, v/v/v/v); stationary phase: upper phase: 900 rpm; 30 C; 300 mg of the EFS: 280 nm; flow rate: 0–100 min, 1.5 mL/min, ∼100 min, (A) 1.5 mL/min, (B) 2.0 mL/min, (C) 2.5 mL/min, (D) 3.0 mL/min. Rs of 1.5 mL/min:67.19%; 2.0 mL/min:65.94%; 2.5 mL/min:64.06%; 3.0 mL/min:62.50%. Journal of Liquid Chromatography & Related Technologies, 38: 1486–1493, 2015 DOI: 10.1080/10826076.2015.1063506 Separation and Purification of Four Flavan-3-ols From Iris Lactea Pall. var. Chinensis (Fisch.) Koidz by High-Speed Counter-Current Chromatography with Flow-Rate Gradient HUANHUAN LV, JIAN OUYANG, XIAOYAN WANG, XIAOFENG MA,2YOURUI SUO, and HONGLUN WANG Flow Rate Gradients
  38. 38. Flow Rate & rpm 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 20 40 60 80 100 120 140 160 r p m flow mL/min flow rate & rpm 0 200 400 600 800 1000 1200 0 1 2 3 4 5 6 r p m flow mL/min flow rate & rpm Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  39. 39. Flow Rate & Volume 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 1 2 3 4 5 6 v o l u m e flow mL/min flow rate & volume 0 2000 4000 6000 8000 10000 12000 14000 16000 0 20 40 60 80 100 120 140 160 v o l u m e flow mL/min flow rate & volume Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  40. 40. Flow Rate/Temperature/Purity Figure 5. Response surface plots for the optimization of HSCCC process. A) Effect of temperature and flow rate on purity of rutin at a constant revolution speed of 850 rpm; Chem. Ind. Chem. Eng. Q. 21 (2) 331−341 (2015) PREPARATIVE ISOLATION AND PURIFICATION OF SEVEN COMPOUNDS FROM Hibiscus mutabilis L. LEAVES BY TWO-STEP HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY ZHUONI HOU, XIANRUI LIAN, FENG SU, WEIKE SU, Figure 3. HSCCC Chromatograms of the first separation. HSCCC Conditions: solvent system: EBuWat (6:1:9 volume ratio); stationary phase: upper phase. Flow rate: 1.11 mL/min, revolution speed: 800 rpm, temperature: 30 °C; stationary phase: upper organic phase; detection wavelength: 254 nm; sample size: 100 mg.
  41. 41. Figure 5. Response surface plots for the optimization of HSCCC process. B) influence of revolution speed and temperature on purity of rutin at a definite flow rate of 1.5 mL/min; Chem. Ind. Chem. Eng. Q. 21 (2) 331−341 (2015) PREPARATIVE ISOLATION AND PURIFICATION OF SEVEN COMPOUNDS FROM Hibiscus mutabilis L. LEAVES BY TWO-STEP HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY ZHUONI HOU, XIANRUI LIAN, FENG SU, WEIKE SU, rpm/Temperature/Purity
  42. 42. Figure 5. Response surface plots for the optimization of HSCCC process. C) interaction between revolution speed and flow rate at a fixed temperature of 25 °C. Chem. Ind. Chem. Eng. Q. 21 (2) 331−341 (2015) PREPARATIVE ISOLATION AND PURIFICATION OF SEVEN COMPOUNDS FROM Hibiscus mutabilis L. LEAVES BY TWO-STEP HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY ZHUONI HOU, XIANRUI LIAN, FENG SU, WEIKE SU, Flow Rate/rpm/Purity
  43. 43. rpm
  44. 44. 0 200 400 600 800 1000 1200 1400 1600 1800 0 500 1000 1500 2000 v o l u m e m L rpm rpm and volume rpm & Volume 0 2000 4000 6000 8000 10000 12000 14000 16000 0 500 1000 1500 2000 v o l u m e m L rpm rpm and volume Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.
  45. 45. Flow Rate & rpm How does the interplay of flow rate and rpm affect K values and resolution? 600 rpm 1.5 mL/mn 800 rpm 1.5 mL/mn 1000 rpm 1.5 mL/mn 600 rpm 2 mL/mn 800 rpm 2 mL/mn 1000 rpm 2 mL/mn 600 rpm 3 mL/mn 800 rpm 3 mL/mn 1000 rpm 3 mL/mn Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  46. 46. 600m rpm 2 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 600 rpm, 1.5 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 600 rpm, 3 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.52 Sf = 0.47 Sf = 0.36 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Increasing Flow Rate Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  47. 47. 600 rpm, 3 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 800 rpm, 3 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.36 Sf = 0.48 1000 rpm, 3 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.55 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Increasing rpm Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  48. 48. 600 rpm, 1.5 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.52 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Increasing Flow Rate & rpm 800 rpm, 2 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.54 2.29 2.67 3.2 4.00 5.33 8.00 16 8 1000 rpm, 3 mL/min 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.55 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  49. 49. Flow Rate & rpm Increasing flow rate increases apparent (experimental) K values and decreases resolution. Especially at lower rpm. Increasing rpm decreases apparent (experimental) K values and increases resolution. Especially at higher rpm. K flow rate rpm Sf
  50. 50. Temperature
  51. 51. Temperature How does temperature affect the K value and resolution? Tauto TBE300A temperature of CS experiments 0 5 10 15 20 20 25 30 35 C numberofarticles 38 data points
  52. 52. 15 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 5 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 10 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.45 Sf = 0.45 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Sf = 0.45 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009) Temperature
  53. 53. 20 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 25 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.50 Sf = 0.54 15 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.45 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009) Temperature
  54. 54. 30 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 35 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 25 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 Sf = 0.53 Sf = 0.57 Sf = 0.54 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009) Temperature
  55. 55. no temperature regulation 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 25 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 A254 30 C 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 Sf = 0.54 Sf = 0.53 Sf = 0.53 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 2.29 2.67 3.2 4.00 5.33 8.00 16 8 Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009) Temperature
  56. 56. Temperature Temperature influences both K values and resolution. Generally, K decreases while Temperature increases. Generally, Temperature influences resolution because compounds respond differently to Temperature changes. Sf as a function of Temperature 0.4 0.5 0.6 0 10 20 30 40degrees C Sf Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  57. 57. Temperature Generally, K tends toward unity while Temperature increases. K Value as a Function of Temperature 0 2 4 6 8 10 1 2 3 4 5 6 7 8 temperature K F U V Q M N E 5 10 15 20 25 30 35room Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)
  58. 58. 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. Temperature Effects Table 2 Partitioning coefficients K of α-carotene and β- carotene and separation factor between them at different temperatures with the two-phase solvent system H/benzotrifluoride/Ac (10:3.5:6.5, v/v/v) determined by HPLC/UV–vis
  59. 59. Parameter Type Parameter Experimental report Essential Important Optional Operational Flow rate E Rpm E Solvent system solvent and volume ratios E Mobile phase identity E Flow direction (head-to-tail, tail-to-head) E Stationary phase volume ratio (Sf) E Switch volume (Vex) of elution extrusion if used E Column equilibration and sample injection method I Temperature I Pressure variation during experiment O Gravitational field generated by rotation O Solvent system phase composition O SS interfacial tension O SS density difference of phases O Viscosity of each phase O pH of aqueous phase O Sample Loading mass of sample E Loading volume E Recovery mass of individual compounds I Enrichment I Composition of active fractions and analytical method I Purity of target analytes and determination method I Partition coefficient (K) of target analytes I Percent recovery of target analytes O Pauli GF, Pro S, Friesen B Countercurrent Separation of Natural Products Journal of Natural Products 71: 1489-1508 (2008) dx.doi.org/10.1021/np800144q Reporting Operational Parameters
  60. 60. Reporting of Separation Parameters: Process Throughput (PT in Grams of Sample Processed per Hour of Separation Time), Process Efficiency (PE in Grams of Sample Processed per Hour of Separation Time), Process Environmental Risk Factor (ER in Liters of Solvent per Gram of Product), Process' General Evaluation Factor (GE in Grams of Sample Times Grams of Product per Hour of Separation Time per Liter of Solvent) natural product(s) (no. of cpds) source solvent system PT [g/h] PE [g/h] ER [l/g] GE [g2 /(h·l)] ref ginsenosides (4) Panax ginseng roots DiMWat and HBuWat (0.1% formic acid) 5.14 0.21–0.54 0.065–0.18 1.17–8.27 140 salvianolic acid B Salvia miltiorrhiza rhizomes HEMWat (0.1% acetic acid) 2.23 0.77 4.0 0.192 250 ginsenosides (4) Panax ginseng roots DiIsoWat (ammonium acetate) 0.23 6.73–14.6 0.06–0.71 9.5–243 251 geniposide Gardenia jasminoides fruits EBuWat 5.0 0.55 4.9 0.113 252 (140) Cheng, Y. J.; Zhang, M.; Liang, Q. L.; Hu, P.; Wang, Y. M.; Jun, F. W.; Luo, G. A. Sep. Purif. Technol. 2011, 77, 347-354. (250) Zhang, M.; Ignatova, S.; Liang, Q. L.; Jun, F. W.; Sutherland, I.; Wang, Y. M.; Luo, G. A. J. Chromatogr. A 2009, 1216, 3869-3873. (251) Qi, X. C.; Ignatova, S.; Luo, G. A.; Liang, Q. L.; Jun, F. W.; Wang, Y. M.; Sutherland, I. J. Chromatogr. A 2010, 1217, 1995-2001. (252) Zhang, M.; Ignatova, S.; Hu, P.; Liang, Q. L.; Wang, Y. M.; Sutherland, I.; Jun, F. W.; Luo, G. A. Sep. Purif. Technol. 2012, 89, 193-198.

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