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Introduction to countercurrent chromatography: instruments

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These slides briefly explain the separation principle and the type of instruments utilized in countercurrent chromatography/ separation.
This workshop presentation was prepared by Dr. Friesen (http://www.dom.edu/departments/physicalsciences/faculty/j-brent-friesen).

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Introduction to countercurrent chromatography: instruments

  1. 1. 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. CounterCurrent Separation Introduction to Instrumentation §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
  3. 3. 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 CCS Cladistic Tree
  4. 4. Partition coefficient (K) Concentration of analyte in one phase/ Concentration of analyte in the other phase shake flask (partitioning) experiment = 4/4 = 1 = 12/36 = 1/3 K = Cupper/Clower
  5. 5. Countercurrent Distribution Series of mixed and equilibrated individual cells All cells contain stationary phase (lower phase in CCD instruments) at the beginning. The first cell is mixed and allowed to settle. The mobile phase is passed to the next cell in the series. New mobile phase is added to the first cell. The first cell contains an equal amount of upper and lower phase plus the sample at the beginning.
  6. 6. 2 3 2 9 2 9 2 27 4 12 4 36 separate phases equilibrate 1 2
  7. 7. 1 1 1 2 2 6 2 12 1½ 7 1½ 14 1 9 1 18 ½ 6 ½ 12 1½ 2.7 1½ 5.3 ½ 0.7 ½ 0.3 4 5
  8. 8. Countercurrent Distribution Lyman C. Craig Sequential countercurrent extractions can separate solutes with only small differences in K. However, the technique, if performed with separatory funnels, is quite tedious. In 1944 Lyman C. Craig developed a device to automate countercurrent distribution. This device used a series of glass vessels. 8 http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1951.tb48879.x/pdf
  9. 9. CCD apparatus takes advantage of successive extractions Countercurrent distribution Jensen 2010, Museum Notes p. 1 Craig CCD Train http://www.erittenhouse.org/wp-content/uploads/2015/07/Fig.-5rs21-973x1030.jpg
  10. 10. CCD output Isopropyl ether and phosphate buffer pH 4.93 http://www.jbc.org/content/174/1/221.full.pdf
  11. 11. CounterCurrent Distribution CCD equations: The rate of migration of the band of a homogeneous substance, that is the position of its maximum, can be calculated from Equation 1 at any time during operation, after a certain number of plates, n, have been applied IDENTIFICATION OF SMALL AMOUNTS OF ORGANIC COMPOUNDS BY DISTRIBUTION STUDIES III. THE USE OF BUFFERS IN COUNTER-CURRENT DISTRIBUTION. BY LYMAN C. CRAIG, CALVIN GOLUMBIC, HAROLD MIGHTON, AND ELWOOD TITUS Journal of Biological Chemistry, 1945, vol. 161, p. 321
  12. 12. http://www.chem.uoa.gr/Applets/AppletCraig/Appl_Craig2.htm 12
  13. 13. Droplet Countercurrent Chromatography http://images.all-free-download.com/images/graphicthumb/different_shapes_water_drop_creative_design_546322.jpg
  14. 14. Droplet Countercurrent Chromatography Eyela DCCC
  15. 15. Droplet Countercurrent Chromatography Ascending mode: lower phase is held stationary in tubes and upper phase bubbled from the bottom. Descending mode: upper phase is held stationary in tubes and lower phase bubbled from the top. continuous flow
  16. 16. Journal of Chromatography A Volume 128, 1976, Pages 218-223 Droplet counter-current chromatography for the separation of plant products Yukio Ogihara, Osamu Inoue Hideaki Otsuka, Ken-Ichi Kawai 1, Takenori Tanimura, Shoji Shibata Droplet Countercurrent Chromatography
  17. 17. Hostettmann, 1980 Droplet Counter-Current Chromatography and its Application to the Preparative Scale Separation of Natural Products 39, p. 1 Droplet Countercurrent Chromatography
  18. 18. Head Tail Correct CorrectX Descending Mode Lower Phase Mobile Ascending Mode Upper Phase Mobile X http://dingo.care2.com/pictures/greenliving/uploads/2013/10/cat-tail.jpg https://cdn.shopify.com/s/files/1/0224/1915/products/grey-tabby-kitty-cat-head-shaped-vinyl-animal-photo-print-clutch-bag-dotoly.jpg?v=1398690617
  19. 19. Centrifugal Partition Chromatography
  20. 20. http://www.labx.co.kr/mall/images/Fg21Dr_FC.gif Centrifugal Partition Chromatography
  21. 21. Centrifuge is used to hold one phase stationary. cells or chambers reminiscent of CCD vessels http://www.labx.co.kr/mall/images/Fg2Dr_FC.gif Centrifugal Partition Chromatography
  22. 22. (High Speed) Countercurrent Chromatography
  23. 23. Continuous Countercurrent Separation in a centrifuge Centrifuge is used to hold one phase stationary. Countercurrent Chromatography http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html under the influence of centrifugal force CounterCurrent Chromatography through the planetary motion of coils (bobbins)
  24. 24. CCS basicsK high K = 1 K low stationary phase mobile phase KD = conc. stationary/conc. mobile phase
  25. 25. CCS basicsK high K = 1 K low
  26. 26. CCS basicsK high K = 1 K low
  27. 27. CCS basicsK high K = 1 K low
  28. 28. CCS basicsK high K = 1 K low
  29. 29. CCS 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 Q HD FUE C V r
  30. 30. Countercurrent Separation Equations VS = volume of stationary phase in the column VM = volume of mobile phase in the column VR = retention volume of analyte K = partition coefficient K = Concentration of analyte in stationary phase Concentration of analyte in mobile phase K = (VR – VM)/VS VC = total column volume = VM + VS
  31. 31. Countercurrent Separation Equations VC = total system volume VS = volume of stationary phase in the column VM = volume of mobile phase in the column VR = retention volume of analyte K = partition coefficient Sf = stationary phase volume retention ratio Sf = VS/VC = (VC – VM)/VC K = (VR – VM)/VS = (VR – VM)/(Sf*VC)
  32. 32. The separation factor (α) is the ratio of K values for 2 consecutive peaks. Countercurrent Separation Equations 𝛼 = 𝐾% 𝐾& 𝐾% > 𝐾& The Resolution factor (RS) s another way to measure the peak separation. W = peak width at the base 𝑅) = %(+, - +.) 0,1 0. http://www.shimadzu.com/an/hplc/support/lib/lctalk/resol-1.html
  33. 33. The theoretical plates (N) is a way to measure chromatographic efficiency. VR and W have the same units. 𝑁 = 16( 𝑉6 𝑊 )% Countercurrent Separation Equations 𝑅) = & 8 𝑁 (+, - +.) :; <= :> 1 ?,@ ?. , 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.
  34. 34. 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. 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
  35. 35. 2 2 1 1 3 3 4 4 5 6 6 8 7 8 7 9 10 9 10 0.5 2 0.5 2 Normal Phase Reversed Phase 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 8 910 6 7 The CS Working Space Non-standardized Standardized Operation Parameters 1. CCC/CPC 2. CCC axes 3. rotational direction 4. synchronicity 5. winding left/right 6. hydrophilic UP/LP 7. hydrophilic VM/VS 8. winding & rotation 9. tail/head 10. K or 1/K Friesen, J.B.; Pauli, G.F. (2009). "Binary concepts and standardization in counter-current separation technology". Journal of Chromatography A 1216 (19): 4237–4244. doi:10.1016/j.chroma.2009.01.048
  36. 36. 7 8 KD = 0.5 0 1 2 Lower (hydrophilic) Phase mobile Head-to-Tail Reversed phase elution 7 9 6 10 0 1 KD =2 Upper (lipophilic) Phase mobile Tail-to-Head Normal phase elution 7 9 6 10 Operation Parameters FWD - IN FWD – OUT REV - IN REV - OUT Lower phase mobile Upper phase mobile HEMWat Solvent System 7 Friesen, J.B.; Pauli, G.F. (2009). "Binary concepts and standardization in counter-current separation technology". Journal of Chromatography A 1216 (19): 4237–4244. doi:10.1016/j.chroma.2009.01.048
  37. 37. Binary Choice Question Answer for example Consequences (6) Aqueous upper or lower? lower Free choice. Any useable biphasic solvent system is appropriate. (8) Do helical winding and rotation agree or oppose? agree Constrained choice. Agreement gives optimal results. (7) Hydrophilic phase mobile or stationary? mobile Free choice. The analyte will elute from the column with either choice. (9) Head-to-tail or tail-to-head? tail-to-head Dependant on (3) The direction of flow must correspond to the choice of mobile phase. (7) Normal or reverse phase? normal phase Dependant on (3) The order of elution will correspond on the choice of mobile phase. (10) Kshake-flask and KCCS; directly or inversely proportional? inversely proportional Dependant on (1) and (3) The order of elution will correspond on the choice of mobile phase. Friesen, J.B.; Pauli, G.F. (2009). "Binary concepts and standardization in counter-current separation technology". Journal of Chromatography A 1216 (19): 4237–4244. doi:10.1016/j.chroma.2009.01.048
  38. 38. Parameter Type Parameter Experimental report Essential Important Optional Instrumental (All) Instrument make, model, and type Column volumea E Sample loop volumea I Extra-column dead volume O Back-pressure regulator setting O Instrumental (type-J specific) Rotor radius (R) I Range of spool radius (r) values I β ratio (βr) I Tubing inside diameter (bore) I Tubing composition I Head center/peripheral O Length of tubing O Number of turns per spool O Direction of winding relative to rotation O Instrumental (CPC specific) Rotor radius I Channel number O Channel volume O Instrumental (Detector) Detector make, model, type E Detector setting (e.g. UV wavelength(s)) E Flow cell details I aAlthough column and sample loop volumes may be given by the manufacturer, they can vary from instrument to instrument and should be measured experimentally and reported as such 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 Instrumentational Parameters

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