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Partitioning experiments

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How to perform partitioning experiments to calculate the partition coefficient K, and ultimately identify or selection the optimal solvent system for countercurrent chromatography.

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Partitioning experiments

  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. Distribution of solute Liquid- Liquid Extraction Miscible in upper phase / Immiscible in lower phase Soluble in both phases more soluble in upper than lower more soluble in lower than upper equally soluble in both Insoluble? Emulsion? Immiscible in upper phase / Miscible in lower phase
  4. 4. Partitioning Experiments Concentration of analyte in one phase/ Concentration of analyte in the other phase Simple extractions Soxhlet Extraction Aqueous Aqueous Aqueous Aqueous n-Butanol Ethyl Acetate Dichloromethane Hexane Kupchan Method 0.271g 0.078g 0.044g 0.563g 3.031g *masses of fractions are based from a starting amount of 20.20g of dandelion root powder
  5. 5. Partitioning Experiments Concentration of analyte in one phase/ Concentration of analyte in the other phase Simple extractions organic/aqueous acid organic/aqueous base
  6. 6. 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
  7. 7. Partitioning Experiment - Analysis HEMWat 1:9:2:8 upper HEMWat 1:9:2:8 lower EGCG ECG EGC Gradient: 60 minutes 5% MeOH /95% Water + 0.05% H3PO4 70% MeOH /30% Water + 0.05% H3PO4 EC
  8. 8. 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 PPM upper and lower phase à 3.7 – 5.5 ppm ECG + EGCG ECG EGCG EC EC + EGC GC C a t EC + EGC EtOAc EGC caffeine GCG GCG HEMWat 1:9:2:8 K= 3.0 1.2 4.5 0.8 EGC K = 0.3 1.2 0.3 0.7 Partitioning Experiment - Analysis
  9. 9. An example of K-by-NMR. The upper and lower-phase samples of HEMWat 4:6:4:6 partition of the ginkgolide enriched sample were subjected to 1H-NMR analysis under the same quantitative experimental conditions. The K values of each terpene lactone were calculated as the ratio of absolute integral of individual H-12 signals in the lower and upper phases. Journal of Chromatography A, 1242 (2012) 26– 34 Design of countercurrent separation of Ginkgo biloba terpene lactones by nuclear magnetic resonance Feng Qiu, J. Brent Friesen, James B. McAlpina, Guido F. Pauli, Partitioning Experiment - Analysis
  10. 10. Standards needed to assign peaks Standards not needed to assign peaks Standards needed for quantification Standards not needed for quantification Sensitivity depends on detector type Sensitivity depends on number of scans 1 -2 mg sample size 20 - 50 mg sample size preferred interference interference “hidden” compounds every H detected solvent does not interfere solvent may interfere peak shifts common minimal peak shifts peak deformation peak deformation chromatographic method development no method development solubility issues solubility issues very small sample size completely non-destructive HPLC vs NMR shakeflask HPLC NMR
  11. 11. Umbelliferone Absorbance values in HEMWat +2 0 0.5 1 260 285 310 335 360 nm A upper phase lower phase K value 2. UV-vis The simple and straightforward “shake-flask” method was used to distribute a commercially available compound between the two phases of a HEMWat system. The concentration of the compound in each phase was measured by its UV/vis absorption or mass. 3. HPLC or GC 1. TLC? 4. qNMR 5. bio-activity Partition coefficients were measured for 22 natural products over the complete range of HEMWat systems. The partition coefficient (K value) of a pure compound is calculated by dividing the concentration of the compound in the upper phase by the concentration of the compound in the lower phase of an equilibrated biphasic system. Partitioning Experiment - Analysis
  12. 12. Partitioning Experiment TLC SS #1 SS #2
  13. 13. TLC Partitioning Analysis He K, Ye X, Li X, Chen H, Yuan L, Deng Y, Chen X, Li X. Separation of two constituents from purple sweet potato by combination of silica gel column and high-speed counter-current chromatography. J Chromatogr B 2012; 881–882: 49–54 “equal volumes (1 μl) of the upper and lower phases were applied on a TLC plate, after separated by the TLC mobile phase, the spot of the target component were scraped from the plate and then extracted to measure the fluorescence intensity using fluorescence spectrophotometer. The K value can be obtained by divide the fluorescence intensity of sample in the upper phase by that in the lower phase.” Separation of PSP extracts by TLC. Mobile phase: chloroform– ethyl acetate (1:1, v/v), 1: 6,7-dimethoxycoumarin, and 2: 5- hydroxymethyl-2-furfural. solvent system: HEMWat (1:2:1:1)
  14. 14. TLC Partitioning Analysis Fig. 1S Selection of solvent systems for HSCCC fractionation of the n-butanol extract of Lantana trifolia. FS = upper phase, FI = lower phase, EBW a) 10:0:10, b) 10:2:10, c) 10:4:10, d) 10:6:10. Planta Med. 2015 Nov;81(17):1609-13. doi: 10.1055/s-0035-1546116. Gradient x Isocratic Elution CCC on the Isolation of Verbascoside and Other Phenylethanoids: Influence of the Complexity of the Matrix. Leitão GG, Pinto SC, de Oliveira DR, Timoteo P, Guimarães MG, Cordova WH, Leitão SG.
  15. 15. TLC Partitioning Analysis Planta Med. 2015 Nov;81(17):1609-13. doi: 10.1055/s-0035-1546116. Gradient x Isocratic Elution CCC on the Isolation of Verbascoside and Other Phenylethanoids: Influence of the Complexity of the Matrix. Leitão GG, Pinto SC, de Oliveira DR, Timoteo P, Guimarães MG, Cordova WH, Leitão SG.
  16. 16. K terminology Partition coefficient Partition ratio Distribution ratio Distribution coefficient Distribution constant Liq-Liq distribution constant Liq-Liq partition ratio K value K K KD Kd Kc KΔ D k k’ http://etc.usf.edu/clipart/81500/81509/81509_menfight_md.gif
  17. 17. Partition coefficient (K) Concentration of analyte in one phase/ Concentration of analyte in the other phase K = Corganic/Caqueous K = Cupper/Clower It is important to define K for partitioning experiments! K = Clower/Cupper K = Caqueous/Corganic
  18. 18. Partition coefficient (K) Concentration of analyte in one phase/ Concentration of analyte in the other phase Solubility of an analyte in one phase/ Solubility of an analyte in the other phase ≈
  19. 19. Partition coefficient (K) Successive extractions of a single phase 100 g of solute in upper phase K = 2/3 = Cupper/Clower 3/5 = 0.6 is the proportion in lower phase 1st extraction 100* 3/5 = 60 grams recovered in lower phase 2nd extraction of upper phase with new lower phase 40*3/5 = 24 grams recovered in lower phase 3nd extraction of upper phase with new lower phase 16*3/5 = 9.6 grams recovered in lower phase
  20. 20. Partition coefficient (K) Successive extractions of a single phase After 3 extractions 60 + 24 + 9.6 = 93.6 g recovered
  21. 21. Partition coefficient (K) http://repository.uobabylon.edu.iq/2010_2011/4_2494_152.pdf

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