Flash: Wide Polarity Range Chromatography

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Wide polarity range chromatography
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  Wide Polarity RangeChromatography Using Combi Flash  ® Rf Systems Abstract In most reactions and subsequent purifications, com-pounds exhibit polarity and solubility properties nearenough to allow purification with a single pair of sol-vents in the gradient. There are times when compoundsof interest span a large polarity range, such as pharma-cognosy applications. By adroit use of gradients andsolvents, chromatograms can be run that span thepolarity range from hexane through water in a single runfor a normal phase column. Reverse phase columns canbe run that span the range from water through methy-lene chloride in a single run. The ability to automaticallychange solvents and gradients saves time and canextend column life. Solid load cartridges avoid solubilityissues since all compounds are released from the car-tridge at the appropriate time in the purification.Examples using a normal phase diol and reversephase C18 columns are presented. Materials and Methods Reverse Phase C18 A crude tomato paste extract was run with a Combi-  Flash ® Rf-200 system equipped with a UV-vis detector(PN 68-5230-008, Teledyne Isco, Lincoln, NE) with a 43 gRedi  Sep  Rf Gold ® C18 column (PN 69-2203-334, TeledyneIsco) using a water to methanol gradient followed by amethanol to methylene chloride gradient with auto-matic solvent switching. A sample was adsorbed ontoCelite ® andplacedintoempty25 gSolidLoadCartridges(PN 69-3873-240, Teledyne Isco). Lycopene compoundswere detected at 473 nm; other compounds were frac-tionated with All Wavelength Collection (200–360 nmrange). The column was initially run with a water/meth-anol gradient which eluted polar compounds thenautomatically switched to a methanol/dichloromethanegradient to purify the lycopenes. This gradient program,includingthesolventchanges,wasprogrammedintotheCombi  Flash  Rf-200 (Figure 1 ). Normal Phase Diol Crude tea extract purification was performed on aCombi  Flash  Rf-200 system (Teledyne Isco, Lincoln, NE,part number 68-5230-006). One gram of extract was dis-solved in methanol and adsorbed onto Celite 545 (AcrosOrganics) in a 5 g Redi  Sep ® solid load sample cartridge(Teledyne Isco part number 69-3873-235). The com-pound was eluted with a hexane/isopropanol gradientfollowed by an isopropanol/water gradient on a 15.5 gRedi  Sep RfGolddiolcolumn(TeledyneIscopartnumber69-2203-515). The multiple solvent gradient was pro-grammed to automatically switch on the Combi  Flash  Rfsystem. The alkaloids and catechin compounds elutedtogether while the tannins eluted early in the water gra-dient (Figure 2 ). Fractions were collected using theAll-Wavelength Collection algorithm. Detection was 254nm (peak width=1 min) and All-Wavelength (200–360nm, peak width=8 min). Results and Discussion Reverse Phase C18 Figure 1: Wide polarity purification of tomatoextract  A1: H 2 O; B1: MeOH; A2: MeOH; B2: DCM Figure 1 demonstrates the ability of the Combi  Flash Rf-200 to automatically switch solvents during purifica-tion and isolate a wide range of compounds in a singlerun. The compounds range from essentially water-sol-uble to completely water insoluble. Normal Phase Diol Figure 2: Initial purification of green tea extract A1: Hexane; B1: IPA; A2: IPA; B2: H 2 O 0473 nmMeOH20 30100       A      b    s    o    r      b    a    n    c    e      %      B      G    r    a      d      i    e    n     t DCM1002Run Time (Column Volumes)All-wavelengthH 2 O MeOH0254 nmhexane20 50100       A      b    s    o    r      b    a    n    c    e      %      B      G    r    a      d      i    e    n     t 1002.5Run Time (Column Volumes)All-wavelength30 40IPA H 2 OIPA Chromatography Application NoteAN77  Chromatography Application Note AN77 Teledyne Isco P.O. Box 82531, Lincoln, Nebraska, 68501 USAToll-free: (800) 775-2965 ã Phone: (402) 464-0231 ã Fax: (402) 465-3001E-mail: IscoService@teledyne.comTeledyne Isco is continually improving its products and reserves the right to change productspecifications, replacement parts, schematics, and instructions without notice. Wide Polarity Range Chromatography works well innormal phase as well (Figure 2 ). In this case, the por-phyrin compounds elute first, followed by catechols andxanthine alkaloids, and finally the tannins at the start ofthe water gradient.For these experiments, one solvent was common toall portions of the gradient. This solvent was defined asB1 and A2 (Figure 3 ); solvent lines A2 and B1 wereplaced in this solvent container. Figure 3: One arrangement of solvents for widepolarity chromatography on C18 After the solvents are configured, the gradient tablecan be generated (Figure 4 ) in the method editor. Figure 4: Programming three solvents for C18 widepolarity range chromatography  running from water through methanol and finishing with dichloromethane. Generally, the B solvent is considered to be the“strong” solvent in chromatography. Keeping the strongsolvent as “B” allows easier conception of the gradient.Figure 5 demonstrates the use of four solvents forwide polarity chromatography. The diol column is ini-tially run with a hexane/ethyl acetate gradient. Thesecond gradient is ethyl acetate/methanol; the final gra-dient is methanol/water. This is a gradient useful forscouting both polar and non-polar compounds for nat-ural products. Figure 5: Four-solvent gradient for wide polarityrange chromatography  on a diol column starting withhexane, going through 100% ethyl acetate to 100% methanoland finishing with water. Conclusion Wide Polarity Range chromatography is an easymethod to purify compounds of widely differing solu-bility with a single run. The use of solid load cartridgesavoid issues of solubility, and avoids compounds precip-itating and clogging the column. The solid load cartridgeallows the full method to be run automatically; the sol-vent changes are programmed into the gradient table ofthe Combi  Flash  Rf-200. The adroit use of solventswitching allows the use of immiscible solvents, such ashexane through ethyl acetate, and then to some concen-tration of methanol on a silica column. CombiFlash, RediSep, and RediSep Rf Gold are registered trademarksof Teledyne Isco, Inc. All other brand names are trademarks of their respective owners. Last modified November 9, 2012  H 2 O MeOHMeOH DCM5 10 15 20 25HexaneH 2 OMeOHEtAc10 20 30 40 50 60
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