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# Reciprocal symmetry plots in Countercurrent Chromatography

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How to represent Countercurrent chromatographic results and what are the Reciprocal Symmetry plots

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### Reciprocal symmetry plots in Countercurrent Chromatography

1. 1. How to ReS and ReSS A 280nm 230nm A K 0 0.125 0.25 0.375 0.5 0.625 0.75 0.875 1 1.14 1.33 1.6 2 2.67 4 8 ∞ IIII II IIIII I1/K GUESSmix in Hexane / Ethyl acetate / Methanol / Water 4:6:4:6 Reverse Phase Normal Phase G H X T r C D F R U V A Q M N Z E O I Y b
2. 2. Traditional Chromatogram HEMWat +2 0 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350mL A 280nm 230nm 120 mL HSCCC (CCC-1000) 1 mL/min, 1,200 rpm fraction collection 3 mL/tube Elution Extrusion Method
3. 3. How to represent the partition coefficient K on the chromatograph? 0 0 50 100 150 200 250 300 350mL A 0 10 20 30 40 50 60 70 80 90 100 280nm 230nm K 0 ≥ K ≤ infinity K = (VR-VM)/VS
4. 4. Reciprocal Symmetry Chose a midline “M” . To the left of “M” à 0 ≥ K ≤ M To the right of “M” à M ≥ K ≤ infinity To graph it: Chose “1” as midline To the left of 1 à x = K To the right of 1 à x = 2-(1/K)
5. 5. Reciprocal Symmetry Plot 0 5 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 X = K Kvalues x = 2- 1/K (0.25,0.25) (1.75,4) ReS Excel plot Reciprocal Symmetry K’(1) adjusted values
6. 6. ReS x-axis adjusted plot Reciprocal Symmetry Plot 0 5 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.25 2 2.5 10 8 K Kvalues 5 Reciprocal Symmetry K axis K = 1/(2-x)
7. 7. Reciprocal Symmetry Chose a midline “M” . To the left of “M” à 0 ≥ K ≤ M To the right of “M” à M ≥ K ≤ infinity To graph it: Chose “M” as midline To the left of “M” à x = K To the right of “M” à x = 2(M)-(M2/K)
8. 8. ReSS Excel Plot Reciprocal Symmetry Plot 0 10 20 30 0 1 2 3 4 5 6 7 8 X = K Kvalues x = 8 – (16/K) (1,1) (7,16) Reciprocal Symmetry K’ adjusted values M = 4
9. 9. ReSS x-axis adjusted plot K = 16/(8-x) Reciprocal Symmetry Plot 0 10 20 30 0 1 2 3 4 8 K Kvalues (1,1) (16,16) 5.33 8 16 Reciprocal Symmetry K axis
10. 10. Chromatograms! HEMWat +2 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2K'(1) A 280nm 230nm 0 0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1) A
11. 11. ReSS HEMWat +2 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4K'(2) A 280nm 230nm 0 0 0.5 1 1.5 2 2.67 4 8 ∞K'(2) A
12. 12. HEMWat +2 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350mL A 280nm 230nm 0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1) A 0 0.5 1 1.5 2 2.67 4 8 ∞K'(2) A T H X r D Y b I OZ M E N A V U F R G Q C
13. 13. How to decide where to put “M”? What looks nice. HEMWat 0 -0.01 0 0.5 1 1.5 2 2.5 3 3.5 4K'(2) A 280nm 230nm HEMWat 0 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2K'(1) A 280nm 230nm
14. 14. M should represent volume midline: Same number of data points on each side of the midline 0 0 0.125 0.25 0.375 0.5 0.625 0.75 0.875 1 1.125 1.25 1.375 1.5 1.625 1.75 1.875 2 A 0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 3.75 4KD A 280nm 230nm G Mix H/EtOAc/MeOH/Water 4:6:4:6 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6K' (3) A 280nm 230nm
15. 15. How to ReS/ReSS What you need: • UV-vis trace for EECCC chromatogram • Time trace for EECCC chromatogram • Mobile phase volumeà VM = void volume • The total time of run à Vrun • Column Volume à VM
16. 16. • Equations: • K = (VR-VM)/(VC-VM) before Vee • K = Vcm/(Vcm+VC-VR) after Vee • ReS x = K’(1) 1 = midline x = K for 0 < K < 1 x = 2-(1/K) for 1 < K < infinity • ReSS x = K’(2) 2 = midline x = K for 0 < K < 2 x = 4-(4/K) for 2 < K < infinity • ReSS x = K’(<M) M = any number as a midline x = K for 0 < K < M x = 2M-(M2/K) for M < K < infinity
17. 17. • Excel Sheet set up: • A – “Elapsed time” in 20 second intervals A2 = date • B – 280nm • C – 230nm • D – mL & min • E – tube (3 mL/tube) • F – VM F2 • Vcm F4 [F8-F10] • Vee F6 [F8-F10+F2] • ` Vrun F8 • VC F10 • G – K Vm < K < Vee [ROUND((D___-F\$2)/(F\$10-F\$2),4)] Vee < K < Vrun [ROUND(F\$4/((F\$4+F\$10)-D___),4)] • H – K’(1) 0 < K < 1 [ROUND((D___-F\$2)/(F\$10-F\$2),4)] 1 < K < infinity [ROUND(2-1/G____,4)] • I – K’(2) 0 < K < 2 [ROUND((D____-F\$2)/(F\$10-F\$2),4)] 2 < K < infinity [ROUND(4-4/G____,4)]
18. 18. Notes With this set-up you can fool around with VM and Vrun to fit the chromatogram nicely into the plot. • Columns G, H, and I require you to manually put the two different equations in for the proper intervals. • Put K values into the plot, for x > midline, once you have it in powerpoint.
19. 19. Applications Instrument comparison Solvent system family mapping Experiment time and separation Reversed-phase / normal phase plot Fractogram ReSS plot
20. 20. GUESSmix in hexane / ethyl acetate / methanol / water 4:6:4:6 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'(2) A 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'(2) A 280nm 230nm J-type centrifuge 120 mL Fast Centrifugal Partition Chromatography (FCPC) 200 mL 220 mg 440 mg G H X T r C D F R U V A Q M N Z E O I Y b
21. 21. HEMWat +3 VCM = 313 mL 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 ∞ KD A 280nm 230nm I II III r C F U V M Q N Z E O b HEMWat +3 VCM = 254.5 mL 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 ∞KD A HEMWat +3 VCM = 228 mL 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 ∞ KD A HEMWat +3 VCM = 162 mL 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 ∞KD A I II III I II III I II III a b c d MS
22. 22. GUESSmix in hexane /ethyl acetate / methanol / water 4:6:4:6 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 mL A 280nm 230nm r C F U V M Q N Z E O b I II III run time = 7.2 hours 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 mL A I II III run time = 6.3 hours Experiment time and separation behavior
23. 23. 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 mL A I II III run time = 4.8 hours 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 mL A I II III run time = 5.8 hours Experiment time and separation behavior
24. 24. A 280nm 230nm A K 0 0.125 0.25 0.375 0.5 0.625 0.75 0.875 1 1.14 1.33 1.6 2 2.67 4 8 ∞ IIII II IIIII I1/K Reversed Phase Normal Phase G H X T r C D F R U V A Q M N Z E O I Y b
25. 25. KD intervals 0 ≤ KD < 0.0625 0.0625 ≤ KD < 0.125 0.125 ≤ KD < 0.25 0.25 ≤ KD < 0.5 0.5 ≤ KD < 1 1 ≤ KD < 2 2 ≤ KD < 4 4 ≤ KD < 8 8 ≤ KD < 16 16 ≤ KD < 32 32 ≤ KD < ∞ HEMWat 0 rXHTG DR CF QUA V N ME Z O I Yb DEMWat 0 rXHT G D C FUV A RQ ZMN E OI Yb GUESS Mix in DEMWat 5:5:5:5 0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1) A HEMWat 0 0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1) A 280nm 230nm O I Yb Z E M NA VU F DRX H T G r C Q I X H T r G D C F U V A R Q ZMNE OIYb
26. 26. Alpinia combined fraction in hexane / tert-butylmethylether / methanol / water 5:5:5:5 0 0.25 0.5 0.75 1 1.33 2 4 ¥K'(1) A Alpinia DCM extract in hexane / ethyl acetate / methanol / water 5:5:5:5 0 0.25 0.5 0.75 1 1.33 2 4 ¥K'(1) A 0 5 10 15 mg 280nm 230nm mg OH O OO HO OO O OH O O OH O
27. 27. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.57 5.33 6.4 8 10.67 16 32 ¥ Oplopanax crude extract in hexane / DCM / methanol / water 7:3:7:3 hexane / ethyl acetate 8:2 hexane / ethyl acetate 7:3 ReS 40 60 80 100 120 140 0 0.25 0.5 0.75 1.33 2 4 ¥K'(1) 40 60 80 100 120 140 0 0.5 1 1.5 2.67 4 8 ¥K'(2) 40 60 80 100 120 140 0 0.5 1 1.5 2 2.5 3.6 4.5 6 9 18 ¥K'(3) 40 60 80 100 120 140 0 1 2 3 5.33 8 16 ¥K'(4) tube tube tubetube ReSS
28. 28. References Reciprocal Symmetry Plots as a New Representation of Countercurrent Chromatograms. G.F. Pauli, J.B. Friesen US 8175817 B2 awarded 05/08/2012. http://www.google.com/patents/US8175817 Friesen, J. B.; Pauli, G. F., Reciprocal symmetry plots as a representation of countercurrent chromatograms. Analytical Chemistry 2007, 79, 2320-2324 Friesen, J. B.; Pauli, G. F., Rational development of solvent system families in counter-current chromatography. Journal of Chromatography A 2007, 1151, 51-59. Friesen, J. B.; Pauli, G. F., Performance characteristics of countercurrent separation in analysis of natural products of agricultural significance. Journal of Agricultural and Food Chemistry 2008, 56, 19-28. Friesen, J. B.; Pauli, G. F., GUESSmix-guided optimization of elution-extrusion counter-current separations. Journal of Chromatography A 2009, 1216, 4225-4231.