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Instrument Methods (Introduction)

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General introduction on the different instrument dependent methods that can be utilized in countercurrent chromatography

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Instrument Methods (Introduction)

  1. 1. (Multiple) Instrument Dependent Methods 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. Multiple Instrument Dependent Methods Scale up See “Optimization Parameters”
  3. 3. Instrument Dependent Methods Sequential CCC
  4. 4. Countercurrent Separations: • Sequential CCS runs CCC Sample Cutting for Isolation of Prenylated Phenolics from Hops Lucas R. Chadwick, Harry H. S. Fong, Norman R. Farnsworth, and Guido F. Pauli Journal of Liquid Chromatography & Related Technologiesw, 28: 1959–1969, 2005
  5. 5. J Chromatogr A. 2007 Jun 1;1151(1-2):169-74. Epub 2007 Jan 11. Advanced applications of counter-current chromatography in the isolation of anti- tuberculosis constituents from Dracaena angustifolia. Case RJ, Wang Y, Franzblau SG, Soejarto DD, Matainaho L, Piskaut P, Pauli GF. Gradient-array counter-current fractionation of D. angustifolia and corresponding anti-TB biochromatogram. Gradient-array CCC fractions from separations using HEMWat 0 (1:1:1:1), HEMWat -3 (3:2:3:2) and HEMWat -5 (7:3:7:3). All fractionation steps utilized elution-extrusion CCC. Resulting fractions are identified as F1–F11. Fractions marked with an X were combined with the A-range from the HEMWat 0 run of the second subsample and reinjected into HEMWat -5. Sequential CCS runs
  6. 6. DCM extract HEMWat 8:2:8:2 HEMWat 5:5:5:5Inactive HEMWat 5:5:5:5 HEMWat 7:3:5:5 HEMWat 7:3:6:4 HEMWat 8:2:8:2 Inactive Compounds 1-7 Compounds 8-15 Compounds 16-23 Compounds 24 -36 Lower phase Lower phase Upper phase Upper phase low K low K low K high K high K high K J Nat Prod. 2010 Apr 23;73(4):563-7. doi: 10.1021/np900674d. Sesquiterpenes from Oplopanax horridus. Inui T1, Wang Y, Nikolic D, Smith DC, Franzblau SG, Pauli GF. Sequential CCS runs
  7. 7. Fig. 2. Scheme of the five countercurrent chromatography (CCC) fractionations which were conducted for the isolation of 20:45,11,14,17 (juniperonic acid) (CCC-1,2,3) and 20:35,11,14 (sciadonic acid) (CCC-1,4,5). Purities (P) and recoveries (R,relative to initial amount) of the target fatty acids as well as the fractions and total mass used for subsequent CCC fractionation are given next to the arrows. CCC-1 HMWat 700:350:4 CCC-2 HAc CCC-3 HMWat 700:350:4 CCC-4 HAc CCC-5 HepMWat 400:364:36 J Chromatogr A. 2015 May 15;1394:89-94. doi: 10.1016/j.chroma.2015.03.042. Isolation of two Δ5 polymethylene interrupted fatty acids from Podocarpus falcatus by countercurrent chromatography. Hammann S, Schröder M, Schmidt C, Vetter W. Sequential CCS runs
  8. 8. HMWat 340:240:1 (1% AgNO3) J Chromatogr A. 2012 May 11;1237:96-105. doi: 10.1016/j.chroma.2012.03.033. Investigation of unsaponifiable matter of plant oils and isolation of eight phytosterols by means of high- speed counter-current chromatography. Schröder M, Vetter W. Sequential CCS runs
  9. 9. J Chromatogr A. 2015 May 15;1394:89-94. doi: 10.1016/j.chroma.2015.03.042. Isolation of two Δ5 polymethylene interrupted fatty acids from Podocarpus falcatus by countercurrent chromatography. Hammann S, Schröder M, Schmidt C, Vetter W. Sequential CCS runs
  10. 10. HterMWat 5:5:2:3 CyterMWat 5:5:2:3 Figure 3. (Top) CCC separation in the first dimension (1D) with the four unresolved compounds a, b, c, and d employing solvent system I. The three heart-cutting time windows are shaded in blue. (Bottom) 2D CCC/UV (254 nm) chromatogram with the corresponding three heart-cuts shaded in green which resolved compounds a, b, c, and d from each other. The elution of the remaining compounds e−h is done in 1D from 156 to 204 min. Anal Chem. 2015 Oct 20;87(20):10172-7. doi: 10.1021/acs.analchem.5b02859. Heart-Cut Two-Dimensional Countercurrent Chromatography with a Single Instrument. Englert M, Brown L, Vetter W.
  11. 11. J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Nov 1; 1004:10-16. doi: 10.1016/j.jchromb.2015.09.017. One-step separation of nine structural analogues from Poria cocos (Schw.) Wolf. via tandem high-speed counter-current chromatography. Zeng H, Liu Q, Yu J, Jiang X, Wu Z, Wang M, Chen M, Chen X. Fig. 2. Chromatograms of T- HSCCC separation. HEMWat (5:5:6:4); stationary phase: upper phase;: 850 rpm; 180 mg: 1.2 mL/min; :25◦C; Sf: 55.7%. Sequential CCS runs
  12. 12. 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. Sequential CCS runs
  13. 13. J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 15;1001:82-9. doi: 10.1016/j.jchromb.2015.07.051. Separation of phenolic acids and flavonoids from Trollius chinensis Bunge by high speed counter-current chromatography. Qin Y, Liang Y, Ren D, Qiu X, Li X. Sequential CCS runs
  14. 14. Peak B 70 mg Peak C 50 mgPetEMWat 1:1:1:1 {0.01 Cu(NO3)2} 180 mg Peak A 60 mg EWat J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 15;1001:58-65. doi: 10.1016/j.jchromb.2015.07.046. Target-guided separation of antioxidants from Semen cassia via off-line two-dimensional high-speed counter-current chromatography combined with complexation and extrusion elution mode. Zeng H1, Liu Q2, Wang M1, Jiang S1, Zhang L1, He X1, Wang J1, Chen X3. Sequential CCS runs
  15. 15. Fig. 2. HSCCC chromatogramsof the n-butanol extract of aerial part of A. ilicifolius, along with the HPLC chromatograms of the crude n-butanol extract and the fractions containing HBOA-Glc (1) and DIBOA-Glc (2) from HSCCC. (B) CCC 1, solvent system: ethyl acetate–n-butanol–0.5%NH4OH (2:3:5, v/v/v); sample, 100mg n-butanol extract dissolved in 8mL of the mixture of ethyl acetate–n-butanol–0.5% NH4OH (2:3:5, v/v/v); (C) CCC 2, solvent system, ethyl acetate–n-butanol–water (2:3:5, v/v/v); sample, 100mg extract dissolved in 8mL of the mixture of ethyl acetate–n-butanol– water (2:3:5, v/v/v); (D) CCC 3, solvent system: ethyl acetate–n-butanol–0.5%NH4OH (2:3:5, v/v/v); sample: peak fraction of compounds 1 and 2 in (C) was evaporated to dryness in vacuo, and dissolved in 8mL of the mixture ethyl acetate–n-butanol–0.5%NH4OH (2:3:5, v/v/v)., 260 mL, 850 rpm, 2.0 mL/min; Sf in (B), (C), and (D) were about 40%, 42%, and 36%, respectively. J Chromatogr A. 2008 Sep 26;1205(1-2):177-81. doi: 10.1016/j.chroma.2008.08.010. Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography. Yin H, Zhang S, Luo X, Liu Y. SequentialCCSruns
  16. 16. Instrument Dependent Methods Column Length
  17. 17. Fig. 2. The representative 1D CCC separations of (a) one, (b) two, (c) three columns connented in series (namely channel A, A–B, A–B–C). 1, dihydrotanshinone I; 2, cryptotanshinone; 3, tanshinone I; 4, 1,2-dihydrotanshinquinone; 5, tanshinone IIA; 6, trijuganone B, 7, methyl tanshinonate, 8, danshenxinkun A. The solvent system of hexane–ethyl acetate–methanol–water was used and prepared on demanded mode: upper phase, hexane:ethyl acetate:methanol:water is 68.70:24.91:3.64:2.75 (v/v, %); lower phase, hexane:ethyl acetate:methanol:water is 2.53:24.77:49.04:23.66 (v/v, %). The sample size was 300 mg and the rotation speed was kept at 500 rpm and flow rate was 3 mL/min. The retention of stationary phase was controlled at 60% by simutaneously pumped the upper phase and lower phase to fill the CCC column, thus the injection mode was injection after equilibrium. Journal of Chromatography A, 1323 (2014) 73– 81 Comprehensive multi-channel multi-dimensional counter-current chromatography for separation of tanshinones from Salvia miltiorrhiza Bunge Jie Meng, Zhi Yang, Junling Liang, Hui Zhou, Shihua Wu,∗ Longer column à Better Resolution
  18. 18. Fig. 4. Separation of caffeine and vanillin with the Arizona L heptane–ethyl acetate–methanol–water 2:3:2:3 (v/v) liquid system. Reversed-phase mode: organic upper stationary phase and aqueous lower mobile phase flown in the head to tail direction. Top chromatograms: column HPCCC Mini 19.6mL with a 0.8mm I.D. tubing coil, 2100 rpm, 2 mL/min. Sf = 38%, Rs = 2.0. Bottom chromatograms: column HPCCC Mini 20.8mL with a 1.6mm I.D. tubing coil, 1800 rpm, 5mL/min, Sf = 66%, Rs = 2.0. Injectionvolume 0.5 mL, concentration 1mg/mL in lower mobile phase. Detection UV at 254 nm. Left chromatograms shown in volume units. Right chromatograms shown in time units. Berthod2009_JCA_1216_4169_SmallVolume HPCCC Mini 19.6mL 0.8 mm tubing HPCCC Mini 20.8mL 1.6 mm tubing Longer column à Better Resolution
  19. 19. Fig. 5. Separation of nine GUESS solutes with HepEMWat 1:1:1:1 Organic upper stationary phase. Top chromatograms obtained in 22min: column HPCCC Mini 19.6mL with a 0.8mmI.D. tubing coil, 2100 rpm, 2 mL/min. Sf = 46%. Bottom chromatograms obtained in 13min: column HPCCC Mini 20.8mL with a 1.6mm I.D. tubing coil,1800 rpm, 5 mL/min, Sf = 72%. Injection volume 0.5 mL, concentration ∼1mg/mL (each) in lower mobile phase. Detection UV at 254 nm. The inset chromatograms are enlargements of the early eluting peaks.. Carvone (O) retention time is indicated. Berthod2009_JCA_1216_4169_SmallVolume HPCCC Mini 19.6mL with 0.8mmI.D. tubing coil HPCCC Mini 20.8mL 1.6mm I.D. tubing Longer column à Better Resolution
  20. 20. Instrument Dependent Methods Sample Prep
  21. 21. Multiple step isolation schemes that integrate CCS Almost a third of the surveyed articles perform a preliminary column chromatography step prior to the CCS experiment. column media: • silica gel • C-18 functionalized silica gel • D101 • XAD-7, XAD-2, XAD-4 • Toyopearl TSK HW-50(F) • AB-8 • Sephadex LH-20 • polyamide preparative steps: § solid-liquid extraction § liquid-liquid extraction § precipitation § flash chromatography

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